Device, Array, And Methods For Disease Detection And Analysis

ABSTRACT

A device and array coupled to capture molecules are provided. Specifically, the device and array can be used for detecting the presence and concentration of biomarkers in a sample from a subject. The device and array can also allow the use of a method for scoring a sample for, e.g., the purpose of diagnosing a disease. The method can also be advantageous to applications where there is a need to accurately determine the disease stage of a subject for the purpose of making therapeutic decisions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/902,147, filed Feb. 15, 2007, the entire disclosure of which ishereby incorporated by reference in its entirety for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The United States Government has rights in this invention pursuant toContract No. DE-AC52-07NA27344 between the United States Department ofEnergy and Lawrence Livermore National Security, LLC for the operationof Lawrence Livermore National Laboratory.

BACKGROUND

1. Field of the Invention

The invention relates to the fields of biology and chemistry.

2. Description of the Related Art

Research over the past decade has focused on discovering new biomarkersthat provide accurate diagnosis of disease, guide therapeutic decisionmaking, and predict the future patterns of disease. Cancer antigen(CA)-125 and Carcinoembryonic antigen (CEA) have both shown promise asbiomarkers for ovarian and colorectal cancer, respectively (Fields M M,C. E. Ovarian cancer screening: a look at the evidence. Clin J OncolNurs. 10, 77-81 (2006); Hasholzner U, S. P., Reiter W, Zimmermann A,Hofmann K, Schalhorn A. CA 242 in comparison with established tumourmarkers in colorectal, pancreatic and lung cancer. Anticancer Res. 19,2477-2480 (1999)). However, while these biomarkers have shown somepotential for possible specific detection of ovarian and colon cancers,no single marker has yet been identified for breast cancer. This can bedue to the fact that breast cancer is not a single disease, but agenetically heterogeneous set of diseases, thus suggesting that it cannot be possible for breast cancer to be diagnosed with any singlemarker. The present invention addresses this need by providing multiple,mutually complementary biomarkers that provide a sensitive diagnosticassay for breast cancer.

Point of care (POC) devices and systems can process samples for a numberof different types of biomarkers in a variety of settings, such asclinical laboratories, patients' bedside and doctors' offices. Variousforms of single biomarker POC technologies are available includingLateral flow assays (LFA) (Panteghini M, P. F. Characterization of arapid immunochromatographic assay for simultaneous detection of highconcentrations of myoglobin and CK-MB in whole blood. Clin Chem ClinBiochem 42, 1292-1293. (1996); Millipore Corp A Short Guide DevelopingImmuno-chromatographic Test Strips. (1996); Lou S C, P. C., Ching S,Gordon J. One-step competitive immunochromatographic assay forsemiquantitative determination of lipoprotein(a) in plasma. J. Clin Chem39 (1993); Lee-Lewandrowski E, L. K. Selected topics in point-of-caretesting—Urinalysis, pregnancy testing, microbiology, fecal occult blood,and other tests. Clin Lab Med 21, 389 (2001)), Disposable microchips(Pugia M J, B. G., Peters R P, Profitt J A, Kadel K, Willms T, Sommer R,Kuo H H, Schulman L S. Microfluidic tool box as technology platform forhand-held diagnostics. Clin Chem. 51, 1923-1932 (2005)), the RAMP™platform (Donald E. Brooks, D. V. D., Paul C. Harris, Joanne E. Harris,Mark E. Miller, Andrew D. Olal, Linda J. Spiller and Zongen C. Xie RAMP:A Rapid, Quantitative Whole Blood Immunochromatographic Platform forPoint-of-Care Testing. Clinical Chemistry 45, 1676-1678 (1999)), and theDual Path Platform (DPP) technology (Carlos Ponce, E. P., ElizabethVinelli, Alberto Montoya, Vilma de Aguilar, Antonio Gonzalez, BiancaZingales, Rafael R. Aldao, Mariano J. Levin, Javan Esfandiari, EufrosinaS. Umezawa, Alejandro O. Luquetti, and José Franco da Silveira.Validation of a rapid reliable test for the diagnosis of Chagas' diseasein blood banks and medical emergencies in Central America. The Journalof Clinical Microbiology, 5065-5068 (2005)). In addition, multiplexedLFAs have also been developed (Jeong D S, C. E. SimultaneousQuantitative Determination of Multiple Analytes with Fluorescence-TaggedProbes by Immunochromatography. Korean J Biol Sci 7, 89-92 (2003)).

Multiplexed LFAs, although sensitive and specific, require elaborateimaging devices for sensitive quantification, thus limiting applicationat the POC. Another form of multiplexed assay are microfabricated flowchannels which pass a sample over an immobilized array (Delehanty J. B.Ligler F. S A microarray immunoassay for simultaneous detection ofproteins and bacteria. Anal. Chem. 74, 5681-5687 (2002); C. R. Taitt, J.P. G., Y. S. Shubin, L. C. Shriver-Lake, K. E. Sapsford3, A. Rasooly, F.S. Ligler A Portable Array Biosensor for Detecting Multiple Analytes inComplex Samples. Microbial Ecology 47, 175-185 (2004); Frances S.Ligler, C. R. T., Lisa C. Shriver-Lake, Kim E, Sapsford, Yura Shubin,Joel P. Golden Array biosensor for detection of toxins. Anal BioanalChem 377, 469-477 (2003); Mark J. Feldstein, J. P. G., Chris A. Rowe,Brian D. MacCraith, Frances S. Ligler Array Biosensor: Optical andFluidics Systems. Journal of Biomedical Microdevices 1, 138-153 (1999)).Use of these assays requires that the sample, detection antibodies, andwash buffers be sequentially introduced at one end of the chamber anddrawn over the microarray surface using a peristaltic pump. These assaysdemonstrate better multiplexed sensitivities as compared to the LFAs,however, they involve sequential detection along the length of the striprather than simultaneous detection, which limits the number ofbiomarkers that can be simultaneously analyzed due to the number ofcapture zones that can be created along the length of the strip. Inaddition, the flow channels are made using polydimethylsiloxane (PDMS)as the material which requires elaborate microfabrication facilities tomanufacture. Also, the fluid exchange through the channels was achievedusing a peristaltic pump, and the assay involved multiple incubation andwash steps, making it challenging to automate and reduce this device toa small, rugged portable form.

The present invention addresses these problems by providing a channelflow device that allows simple, rapid, and sensitive detection ofmultiple biomarkers.

SUMMARY

Disclosed herein is a detection device. In one aspect, the detectiondevice includes a solid support including a plurality of distinctcapture molecule groups, each distinct capture molecule group includinga plurality of capture molecules specific for a biomarker, wherein theplurality of distinct capture molecule groups is specific for aplurality of biomarkers; a cover plate, wherein the cover plate forms anupper surface positioned above the solid support; a vertical support,wherein the vertical support forms a connection between the solidsupport and the cover plate, the connection forming at least one channelaround the capture molecule groups, and wherein the channel includes afirst end and a second end and wherein the first end of the channelincludes an opening; and an absorbent material connected to the secondend. In another aspect of the detection device, the solid supportincludes glass. In another aspect of the detection device the solidsupport includes a glass slide.

In one embodiment, the capture molecules include antibodies. In anotherembodiment, the capture molecules are specific for biomarkers selectedfrom Her-2, MMP-2, CA 15-3, VEGF, and OPN. In another embodiment, thecapture molecules are specific for Her-2, MMP-2, CA 15-3, VEGF, OPN,p53, CA 125, and SER. In another embodiment, the capture molecules arespecific for Her-2, MMP-2, CA 15-3, and OPN. In another embodiment, thecapture molecules are Clone 191924, Clone 36006.211, Clone M8071022, andClone 190312. In another embodiment, the capture molecules are blockedby a blocking agent. In another embodiment, the plurality of distinctgroups of capture molecules is arranged in an array format. In anotherembodiment, the solid support includes at least two capture moleculegroups including identical capture molecules, and each of the at leasttwo capture molecule groups including a different number of capturemolecules.

In one embodiment, the cover plate includes glass. In anotherembodiment, the cover plate is a glass cover slip. In one embodiment,the vertical support includes adhesive silicone. In one embodiment, theabsorbent material includes a Hi-Flow Plus Nitrocellulose MembraneHF240.

In another aspect, the detection device includes a glass slide includingan array of a plurality of distinct groups of antibodies cross-linked tothe slide, each distinct group specific for a biomarker selected fromHer-2, MMP-2, CA 15-3, and OPN; a glass cover slip positioned above thesolid support; and a silicone adhesive connection between the glassslide and the glass cover slip forming at least one channel around theantibody groups, and wherein the channel includes a first open end and asecond end connected to a Hi-Flow Plus Nitrocellulose Membrane HF240.

In one embodiment, the detection device further includes a component fordetecting biomarkers bound to the solid support. In a relatedembodiment, the component includes an optical reader and a screen fordisplaying output from the optical reader.

Also disclosed herein is a method for determining the presence orabsence of a plurality of biomarkers in a sample, including: acquiring aliquid mixture, wherein the mixture includes the sample; applying themixture to the open first end of the at least one channel of the device,described above; allowing the mixture to flow through the at least onechannel over the solid support; absorbing the mixture with the absorbentmaterial connected to the second end; and detecting the presence ofbiomarkers on the solid support, wherein presence of the biomarkers onthe solid support indicates the presence of the biomarkers in thesample.

In one embodiment of the method for determining the presence or absenceof a plurality of biomarkers in a sample, the detection device describedabove, is used, the device includes capture molecules includingantibodies and the liquid mixture includes the sample, at least onedetector antibody, and at least one fluorescent reporter, and the methodfurther including the steps of analyzing the sample with an opticalreader to determine the presence or absence of the plurality ofbiomarkers in the sample; and outputting the data, wherein the datainclude the presence or absence of the plurality of biomarkers in thesample. In another embodiment of the method for determining the presenceor absence of a plurality of biomarkers in a sample, the detectiondevice, described above, includes capture molecules specific for aplurality of biomarkers selected from the group consisting of CA 15-3,OPN, Her-2, and MMP-2. In another embodiment, the sample includes humanblood serum.

Also disclosed herein is an array of antibodies immobilized on a solidsupport, the array including: a plurality of distinct antibody groups,each distinct antibody group including a plurality of antibodiesspecific for a biomarker, wherein the plurality of distinct antibodygroups is specific for a plurality of biomarkers, and wherein theplurality of biomarkers include CA 15-3, OPN, Her-2, and MMP-2.

Also disclosed herein is a method for determining protein concentrationdata in a sample with an array, including: acquiring a mixture, whereinthe mixture is in a liquid state, and wherein the mixture includes thesample from a mammalian subject, a detector antibody, and a fluorescentreporter; applying the mixture to the array described above; analyzingthe sample with a reader to determine the concentration of the pluralityof biomarkers in the sample; and outputting the data, wherein the datainclude protein concentration data for the plurality of biomarkers, andwherein the plurality of biomarkers include CA 15-3, OPN, Her-2, andMMP-2.

Also disclosed herein is a method of scoring a sample acquired from amammalian subject, including: obtaining a first dataset includingquantitative data associated with a plurality of biomarkers associatedwith breast disease and the plurality of biomarkers include CA 15-3, andOPN, wherein the data include measured values obtained from the sample;analyzing the first dataset against a second dataset to produce a scorefor the sample; and outputting the score.

In one embodiment, the plurality of biomarkers includes Her-2. Inanother embodiment, the plurality of biomarkers includes MMP-2. Inanother embodiment, the plurality of biomarkers includes Her-2 andMMP-2. In another embodiment, the plurality of biomarkers includesHer-2, MMP-2, VEGF, p53, CA 125, and SER. In another embodiment, thequantitative data includes protein concentrations. In anotherembodiment, the data is immunoassay data. In another embodiment, theprotein concentrations are obtained using an immunoassay includingantibodies. In a related embodiment, the immunoassay is a sandwichimmunoassay. In another embodiment, the protein concentrations areobtained using a multiplexed channel flow-based device. In anotherembodiment, the antibodies of the immunoassay are Clone 191924, Clone36006.211, Clone M8071022, Clone 190312, and Clone A183C-13G8.

In another embodiment, the analyzing step includes use of a predictivemodel. In a related embodiment, the predictive model is developed usingprincipal component analysis. In another related embodiment, thepredictive model is developed using linear discriminant analysis. Inanother embodiment, the analyzing step includes categorizing the sampleinto categories according to a score produced with the predictive model.In a related embodiment, the categorization is selected from the groupconsisting of: a healthy categorization, an early-stage diseasecategorization, and a late-stage disease categorization. In anotherrelated embodiment, a probability that the categorization is correct isat least 60%, at least 70%, at least 80%, at least 87%, at least 90%,and at least 95%.

In another embodiment, the method further includes comparing the scoreto a second score determined for a second sample obtained from themammalian subject. In a related embodiment, wherein a difference betweenthe first score and the second score indicates a disease stage of breastcancer. In another embodiment, wherein the mammalian subject is a humansubject. In another embodiment, wherein the score is used to diagnose aneoplastic breast disease. In another embodiment, wherein the breastdisease is breast cancer.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, and accompanying drawings, where:

FIG. 1 is a schematic representation of the point of care channelflow-based immunoassay detection device.

FIG. 2 is a schematic of the benchtop imaging system.

FIG. 3 shows testing for the specificity of the tested antibodies usingwestern blotting with five antigens.

FIG. 4 shows optimization of capture and detector antibodyconcentrations for developing standard curves using enzyme linkedimmunosorbent assay (ELISA).

FIG. 5 are standard curves using ELISA.

FIG. 6 is a schematic of the sandwich immunoassay format used in proteinmicroarrays.

FIG. 7 shows optimization of capture and detector antibodyconcentrations for developing standard curves using the antibodymicroarray immunoassay.

FIG. 8 is visualization of standard curves using the antibodymicroarrays.

FIG. 9 is a comparison of standard curves obtained in a single plex anda multiplex format. using the antibody microarrays, with phosphatebuffered saline (PBS) and Serum as the medium.

FIG. 10 is a comparison of standard curves obtained using antibodymicroarrays with the ELISA.

FIG. 11 shows multiplexed assays on protein microarrays.

FIG. 12 shows the experimental layout of the breast cancer sample pilotstudy.

FIG. 13 shows the pilot studies with 30 breast cancer patient samples.

FIG. 14 shows the experimental layout of the breast cancer study.

FIG. 15 shows the measurement of biomarkers in breast cancer patientsera.

FIG. 16 is the standard curves for biomarkers in multiplex format.

FIG. 17 shows clustering and linearization of the four dimensional dataobtained using the multiplex assay and principle component analysis.

FIG. 18 shows classification error estimate using linear discriminantanalysis; H=Her-2; M=MMP-2; O=OPN; C=CA 15-3.

FIG. 19 is a schematic representation of a lateral flow test strip.

FIG. 20 shows fluorescence images of the lateral flow assay (LFA).

FIG. 21 shows fluorescence images of the sandwich LFA.

FIG. 22 shows the results of the proof of principle on microarraychannels.

FIG. 23 shows standard curves using Quantum Dots on microarray channels.

FIG. 24 shows the multiplex assay using Quantum Dots on microarraychannels.

FIG. 25 shows the results of troubleshooting the Quantum Dot assay onmicroarray channels.

FIG. 26 is the standard curves obtained using microarray channels in 15min.

FIG. 27 is a demonstration of multiplexed immunoassays on microarrayflow channels.

FIG. 28 is a determination of the best combination of assay speed andsensitivity for the microarray flow channels.

FIG. 29 shows biomarker concentration in patient serum samples.

FIG. 30 shows imaging system standard curves.

FIG. 31 is a comparison of the imaging system and a photomultiplier tube(PMT).

DETAILED DESCRIPTION

Briefly, and as described in more detail below, described herein is achannel flow based immunoassay detection device for determining thepresence and/or concentration of a plurality of biomarkers in a sample.Also disclosed are methods of using the device, and arrays of capturemolecules, e.g., antibodies, for use with the device. In one embodiment,the device is used to detect a plurality of biomarkers related to breastcancer. Described herein are methods of scoring a sample using dataassociated with the breast cancer biomarkers.

Advantages of this approach are numerous. The device provides theability to perform multiplexed analysis of multiple biomarkers in aformat that is simple to use, amenable to automation, and in a small,rugged format. The device has been developed in a point of care (POC)format, allowing for rapid diagnostic assay, and facilitating fastertherapeutic decisions and possible increased patient survival rates.

The device can be used to diagnose and prescribe treatment for a widevariety of medical conditions, especially cancers, heart diseases,respiratory diseases, and microbial infections. In one embodiment, thedevice is used to diagnose breast cancer.

Also disclosed is a multiplexed immunoassay to detect a set ofbiomarkers associated with breast cancer. The immunoassay can accuratelydetect a panel of two, three, four, five, six, seven, or eightbiomarkers from the sera of breast cancer patients and distinguishbetween control, early stage, and metastatic breast cancer populations.The immunoassay was shown to predict the stage of unknown sample. Theassay can be used can be used along with mammography for resultvalidation and in between annual mammograms to diagnose rapidly-growingtumors. The advantage of the multiplex assay is the ability to determinethe levels of these markers simultaneously, thus reducing time, effort,overall volume of reagent and patient sample. Such a panel can offer acomplete range of tests such as diagnosis, prognosis, treatment optionsand treatment monitoring in a single assay, providing additionalinformation enabling rapid diagnosis and improved patient survivalrates.

DEFINITIONS

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified.

A “capture molecule” is a molecule that is immobilized on a surface. Thecapture molecule generally, but not necessarily, binds to a target ortarget molecule, e.g., a biomarker. The capture molecule is typically anantibody, a peptide, or a protein. In the case of a solid-phaseimmunoassay, the capture molecule is immobilized on the surface of asolid support and is an antibody specific to the target, an antigen orepitope, to be detected. The capture molecule can be labeled, e.g., afluorescently labeled antibody or protein. The capture molecule can orcan not be capable of binding to just the target. Capture molecules caninclude e.g., RNA, DNA, peptides, antibodies, aptamers, andprotein-based aptamers. In one embodiment the capture molecule is anantibody.

A “biomarker” is a molecule of interest that is to be detected and/oranalyzed, e.g., a peptide, or a protein. Typically a biomarker isassociated with a particular physical condition, e.g., a disease ordisease state, e.g., late stage breast cancer.

A biomarker that “binds” to a capture molecule is a term well understoodin the art, and methods to determine such specific or preferentialbinding are also well known in the art. A molecule is said to exhibit“binding” if it reacts or associates more frequently, more rapidly, withgreater duration and/or with greater affinity with a particular targetthan it does with alternative substances. A capture molecule “binds” toa target if it attaches with greater affinity, avidity, more readily,and/or with greater duration than it attaches to other substances. Forexample, a capture molecule that specifically or preferentially binds toa target is an antibody that binds this target with greater affinity,avidity, more readily, and/or with greater duration than it binds toother substances. It is also understood by reading this definition that,for example, a capture molecule that specifically or preferentiallybinds to a first target may or may not specifically or preferentiallybind to a second target. As such, “binding” does not necessarily require(although it can include) exclusive binding. Generally, but notnecessarily, reference to “binding” means preferential binding. Theconcept of “binding” also is understood by those of skill in the art toinclude the concept of specificity. Specific binding can bebiochemically characterized as being saturable, and binding for specificbinding sites can be biochemically shown to be competed.

An “antibody” is an immunoglobulin molecule capable of specific bindingto a target, such as a carbohydrate, polynucleotide, lipid, polypeptide,etc., through at least one antigen recognition site, located in thevariable region of the immunoglobulin molecule.

The terms “polypeptide,” “oligopeptide,” “peptide,” and “protein” areused interchangeably herein to refer to polymers of amino acids of anylength. The polymer can be linear or branched, it can comprise modifiedamino acids, and it can be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), as well as other modifications known in the art.

An “array” format is a known or predetermined and ordered spatialarrangement of one or more capture molecules on a solid support. A“multiplexed array” format is an ordered spatial arrangement of two ormore capture molecules on a solid support. In one embodiment, row andcolumn arrangements are used due to the relative simplicity in makingand assessing such arrangements. The spatial arrangement can, however,be essentially any form selected by the user, and preferably, but neednot be, in a pattern. Array formats are characterized by the use ofspatial location within the array to identify the feature present atthat location.

“Detect” refers to identifying the presence, absence and/or amount ofprotein to be detected. Detection can be done visually or using adevice, e.g., a scanner and detector.

The term “mammal” as used herein includes both humans and non-humans andinclude but is not limited to humans, non-human primates, canines,felines, murines, bovines, equines, and porcines.

“Solid support” refers to a material or group of materials having arigid or semi-rigid surface or surfaces. In some aspects, at least onesurface of the solid support will be substantially flat, although insome aspects it can be desirable to physically separate regions fordifferent molecules with, for example, wells, raised regions, pins,etched trenches, or the like.

To “analyze” includes determining a set of values associated with asample by measurement of constituent expression levels in the sample andcomparing the levels against constituent levels in a sample or set ofsamples from the same subject or other subject(s).

A “predictive model” is a mathematical construct developed using analgorithm or algorithms for grouping sets of data to allowdiscrimination of the grouped data. As will be apparent to one ofordinary skill in the art, a predictive model can be developed usinge.g., principal component analysis (PCA), and linear discriminantanalysis (LDA).

A “score” is a value or set of values selected or used to discriminate asubject's condition based on, for example, a measured amount of sampleconstituent from the subject.

The term percent “identity,” in the context of two or more nucleic acidor polypeptide sequences, refers to two or more sequences orsubsequences that have a specified percentage of nucleotides or aminoacid residues that are the same, when compared and aligned for maximumcorrespondence, as measured using one of the sequence comparisonalgorithms described below (e.g., BLASTP and BLASTN or other algorithmsavailable to persons of skill) or by visual inspection. Depending on theapplication, the percent “identity” can exist over a region of thesequence being compared, e.g., over a functional domain, or,alternatively, exist over the full length of the two sequences to becompared.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information, most conveniently at itswebsite.

Abbreviations used include: human estrogen receptor-2 (Her-2), matrixmetallopeptidase-2 (MMP-2), cancer antigen 15-3 (CA 15-3), osteopontin(OPN), tumor protein 53 (p53), vascular endothelial growth factor(VEGF), cancer antigen 125 (CA 125), Serum Estrogen Receptor (SER)

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise.

Devices of the Invention

The invention provides a POC, multiplexed, channel flow-basedimmunoassay detection device as shown in FIG. 1. The device is comprisedof a solid support, a cover plate, a vertical support, and an absorbentmaterial. The solid support has an array of capture molecules, e.g.,antibodies specific for set of biomarkers, immobilized on its uppersurface. One or more flow channels are formed by coupling the uppersurface of the solid support to the lower surface of the cover platewith the vertical support. Coupling is performed using, e.g., anadhesive. When coupled with the vertical support, the device has an openend for administration of a fluid mixture and a closed end opposite ofthe open end that is closed with the absorbent material. The flowchannel is designed in a manner that allows for flow of the fluidmixture over the immobilized array of capture molecules and that ispreferably driven by capillary action, although in certain embodimentsinvolving relatively larger sample volumes, bulk flow forces may operatethe device.

The fluid mixture is typically a sample, e.g., a blood sample thatincludes the biomarkers of interest. Often the fluid mixture includesadditional reagents, e.g., detection antibodies, for detection of thebiomarkers bound to the capture molecules immobilized on the solidsupport. The detection antibodies can be labeled for detection, e.g.,fluorescently labeled.

In use, the fluid mixture is added to the open end of the device. Thisfluid is drawn into the channel and over the immobilized array by forcepreferably produced by capillary action. The fluid is then wicked fromthe opposite end of the channel by the absorbent material. Flow throughthe flow channel is unidirectional due to the absorbent material.Protein biomarkers in the fluid mixture that are bound by the capturemolecules are quantified, e.g., by an optical reader that detects thefluorescently labeled detection antibodies bound to the biomarkers boundto the capture molecules immobilized to the array. The fluorescence ofthe array is proportional to the biomarker concentration in the fluidmixture.

Solid Support

The device includes a solid support comprising immobilized capturemolecules, e.g., antibodies. Solid supports suitable for immobilizing,binding and/or linking antibodies (and modifications to render solidsupports suitable for immobilizing capture molecules) are well known inthe art. Examples of a solid support include: a microwell plate and aprotein microarray (e.g., technology owned by Zyomyx, Inc. See, e.g.U.S. Pat. No. 6,365,418). In addition, pads, film, nanowells, ormicrofluid channels can also serve as a solid support. In someembodiments, the capture molecules are immobilized, bound, or linked ona solid support surface such as polyvinylidene difluoride,nitrocellulose, agarose, and/or polyacrylamide gel pads. In otherembodiments, the solid support can be made of glass or include a glassslide. Glass slides activated with aldehyde, polylysine, or ahomofunctional cross-linker can also been used. In yet otherembodiments, the capture molecules can be arranged in athree-dimensional array, for example in the three dimensionalpolyacrylamide gel pad microarray described in Mirzabekov et al.,Nucleic Acids Res 24(15): 2998-3004 (1996).

The invention provides a solid support, wherein capture molecules areimmobilized. For the purposes of the invention, the term “immobilized”includes immobilized, bound, or linked to the solid support. Linking canbe covalent or noncovalent. Methods of linking capture molecules to thesolid support are well known in the art. See, e.g. Kennedy et al. (Clin.Chim. Acta 70:1-31 (1976)), and Schurs et al. (Clin. Chim. Acta 81:1-40(1977)) (describing coupling techniques, including the glutaraldehydemethod, the periodate method, the dimaleimide method, them-maleimidobenzyl-N-hydroxy-succinimide ester method, all of whichmethods are incorporated by reference herein).

The capture molecules can be bound to many different solid supportmaterials. Examples of well-known materials include polypropylene,polystyrene, polyethylene, polymers, dextran, nylon, amylases, glass,natural and modified celluloses, polyacrylamides, agaroses, silicone,and magnetite. Other materials are well known in the art. See, e.g.,Angenendra et al., Next generation of protein microarray supportmaterials: Evaluation for protein and antibody microarray applications;Journal of Chromatography A; Volume 1009, Issues 1-2, 15 Aug. 2003,Pages 97-104.

Preferably, the capture molecules are arranged on the solid support inan array format. More preferably, the capture molecules are arranged onthe solid support in a multiplex array format. Alternatively, thecapture molecules can be arranged on the solid support in ordered,sequential lines. Capture molecules and detection agents, includingsuitable labels, are further described herein.

Capture Molecules

The invention further provides a plurality or set of capture molecules,wherein the set comprise at least about 2 distinct capture molecules,wherein each distinct capture molecule recognizes a different biomarker,e.g., peptide or target. In some embodiments, the set comprises at leastabout 3, 4, 5, 6, 7, 8, or more distinct capture molecules.

A capture molecule can encompass monoclonal antibodies, polyclonalantibodies, antibody fragments (e.g., Fab, Fab′, F(ab′)₂, Fv, Fc, etc.),chimeric antibodies, single chain Fvs (ScFvs), mutants thereof, fusionproteins comprising an antibody portion, and any other polypeptide thatcomprises an antigen recognition site of the required specificity(including antibody mimetics. See, e.g., Xu et al, Chem. Biol. 2002 Aug.9(8):933-42). The antibodies can be murine, rat, rabbit, chicken, human,or any other origin, including humanized antibodies. Capture molecules,such as antibodies, can be made recombinantly and expressed using anymethod now known or later discovered in the art. In addition, antibodiescan be made recombinantly by phage display technology. For examples ofthese expression and production methods see e.g., U.S. Pat. Nos.5,565,332; 5,580,717; 5,733,743 and 6,265,150; and Winter et al., Annu.Rev. Immunol. 12:433-455 (1994).

As used herein, the term “antibody” encompasses not only intactpolyclonal or monoclonal antibodies, but also fragments thereof (such asFab, Fab′, F(ab′)₂, Fv), single chain (ScFv), mutants thereof, fusionproteins comprising an antibody portion, and any other modifiedconfiguration of the immunoglobulin molecule that comprises an antigenrecognition site of the required specificity. An antibody includes anantibody of any class, such as IgG, IgA, or IgM (or sub-class thereof),and the antibody need not be of any particular class. Depending on theantibody amino acid sequence of the constant domain of its heavy chains,immunoglobulins can be assigned to different classes. There are fivemajor classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, andseveral of these can be further divided into subclasses (isotypes),e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constantdomains that correspond to the different classes of immunoglobulins arecalled alpha, delta, epsilon, gamma, and mu, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

“Fv” is an antibody fragment that contains a completeantigen-recognition and -binding site. In a two-chain Fv species, thisregion consists of a dimer of one heavy and one light chain variabledomain in tight, non-covalent association. In a single-chain Fv species,one heavy and one light chain variable domain can be covalently linkedby a flexible polypeptide linker such that the light and heavy chainscan associate in a dimeric structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three complementaritydetermining regions (CDRs) of each variable domain interact to define anantigen-binding specificity on the surface of the VH-VL dimer. However,even a single variable domain (or half of a Fv comprising only 3 CDRsspecific for an antigen) has the ability to recognize and bind antigen,although generally at a lower affinity than the entire binding site.

A “monoclonal antibody” refers to a homogeneous antibody populationwherein the monoclonal antibody is comprised of amino acids (naturallyoccurring and non-naturally occurring) that are involved in theselective binding of an antigen. A population of monoclonal antibodies(as opposed to polyclonal antibodies) are highly specific, in the sensethat they are directed against a single antigenic site. The term“monoclonal antibody” encompasses not only intact monoclonal antibodiesand full-length monoclonal antibodies, but also fragments thereof (suchas Fab, Fab′, F(ab′)₂, Fv), single chain (ScFv), mutants thereof, fusionproteins comprising an antibody portion, and any other modifiedconfiguration of the immunoglobulin molecule that comprises an antigenrecognition site of the required specificity and the ability to bind toan antigen (see definition of antibody). It is not intended to belimited as regards to the source of the antibody or the manner in whichit is made (e.g., by hybridoma, phage selection, recombinant expression,transgenic animals, etc.).

Capture molecules can be blocked using blocking agents such as, e.g.,serum or serum diluted in phosphate buffered saline (PBS) and otherblocking agents known in the art.

The choice of capture molecules depends on the application and thebiomarkers to be detected in the sample. In one embodiment, thebiomarkers to be detected are associated with breast cancer and include,e.g., include Her-2, MMP-2, CA 15-3, OPN, p53, VEGF, CA 125, and SerumEstrogen Receptor (SER). These biomarkers can include known fragments,splice variants, and full length peptides as well as other variationsthat are not currently known. Examples of sequence identifiers as ofFeb. 11, 2008 at the HUGO on-line database for these markers include,but are not limited to, Her-2 (X03363), MMP-2 (NM_(—)004530), OPN(NM_(—)001040058), p53 (NM_(—)000546), VEGF (MGC70609), CA 125 (Q8WX17),SER (NP_(—)000116.2), and CA 15-3 (NM_(—)002456). Additional sets ofbiomarkers can be chosen for other diseases including prostate cancer,ovarian cancer, and heart disease. Biomarker targets for prostate cancercan include prostate specific antigen (PSA). Biomarker targets forovarian cancer can include CA 125. Biomarker targets for heart diseasecan include Troponin T, Troponin I, C-reactive protein (CRP),Homocysteine, Myoblobin, and Creatine kinase. In addition, capturemolecules specific for biomarker associated with respiratory diseasescan be chosen, including biomarkers associated with influenza A,influenza B, Anthrax, Plague, and allergens.

In one embodiment, the capture molecules are capture antibodies specificfor breast cancer markers. Capture antibodies specific for breast cancermarkers can include: 1) anti-Her-2 (R&D systems; Monoclonal Anti-humanErbB2 Antibody; MAB-1129; Clone 191924), 2) anti-Matrix metallopeptidase(MMP)-2 (R&D systems; Monoclonal Anti-human MMP-2 Antibody; MAB-902;Clone 36006.211), 3) anti-CA 15-3 (Fitzgerald; Monoclonal Anti-human CA15-3 Antibody; 10-C03; Clone M8071022), 4) anti-Osteopontin (OPN) (R & DSystems; Monoclonal Anti-human Osteopontin Antibody; MAB-1433; Clone190312), and 5) anti-Vascular Endothelial Growth Factor (VEGF)(Biosource; VEGF purified mouse anti-human; AHG011; Clone A183C-13G8).

As described herein, a capture molecule can bind a peptide epitope of 2or more consecutive (i.e., sequential) amino acids. It is understoodthat the amino acid(s) forming the target epitope can be linear orbranched, and can comprise an amino acid(s) that has been modifiednaturally or by intervention; for example, disulfide bond formation,glycosylation, lipidation, acetylation, phosphorylation, or any othermanipulation or modification, such as conjugation with a labelingcomponent. The amino acid(s) forming the target epitope can furtherencompass, for example, one or more analogs of an amino acid (including,for example, unnatural amino acids, etc.), as well as othermodifications known in the art. In some embodiments the target is aprotein biomarker. Protein biomarkers are further described herein.

In some embodiments, the capture molecule binds its cognate targetepitope with an affinity of binding reaction of at least about 10⁻⁷ M,at least 10⁻⁸ M, or at least about 10⁻⁹ M, or tighter. In someembodiments, a binding interaction will discriminate over adventitiousbinding interactions in the reaction by at least two-fold, at leastfive-fold, at least 10- to at least 100-fold or more.

It is understood that other target binding agents can be used, inaddition to the capture molecule sets described herein. In addition, itis evident that the number of capture molecules in the capture moleculeset depends on the contemplated uses and applications of the set,complexity of the sample, average size of the proteins in the sample,frequency that the cognate target epitope is present or predicted to bepresent in a sample, binding affinity and/or specificity of the capturemolecules, knowledge of target protein(s), and stability of the capturemolecules. Such factors, and others, are well known in the art.

Detection of Biomarkers

Detection of target, e.g., biomarker, binding to a capture molecule canbe made using detection molecules and detection agents. In oneembodiment, detection molecules are detection antibodies. Detectionantibodies are specific for a biomarker target, similar to captureantibodies or capture molecules.

Detection agents can be labeled using methods well-known to one of skillin the art, e.g., detection agents can be fluorescent agents,colorimetric agents, and magnetic agents. Fluorescent agents can includee.g., quantum dots and fluorophores, e.g., ALEXA 546.

In one embodiment, the detection antibodies include: 1) anti-Her-2 (R &D Systems; Polyclonal Goat Anti-human ErbB2 Antibody; AF-1129), 2)anti-MMP-2 (R & D Systems; Polyclonal Goat Anti-human MMP-2 Antibody;AF-902), 3) anti-CA 15-3 (Fitzgerald; Monoclonal Anti-human CA 15-3antibody; 10-C03B; Clone M8071021), 4) anti-Osteopontin (R & D Systems;Polyclonal Goat Anti-human Osteopontin Antibody; AF-1433), and 5)anti-VEGF (Biosource; Polyclonal Rabbit Anti-human VEGF BiotinConjugated Antibody; AHG9119).

Cover Plates

The device includes a cover plate coupled to the upper surface of thesolid support via vertical supports. The cover plate provides an uppersurface of the channel through which fluid flows. In some embodiments,the cover plate is positioned a fixed distance above the solid supportfor allowing the entrance of fluids into a channel formed by thecoupling. The cover plate can be made of any material suitable for theapplication, e.g., polypropylene, polystyrene, polyethylene, polymers,dextran, nylon, plastic, amylase, silicone, glass, natural and modifiedcelluloses, polyacrylamides, agaroses, and magnetite. In one embodiment,the cover plates are made of a hydrophilic material. In anotherembodiment, the cover plates are made of an optically transparentmaterial.

In another embodiment, the cover plate is a cover slip made of glass. Inanother embodiment, the cover plate is a Corning® cover glass rectangle,Cat # 2935-244 size 24 mm (W)×40 mm (H)×0.13 mm (Thick).

Vertical Supports

The cover plate is connected to the solid support using a verticalsupport. The vertical support can be singular or, the device can includea plurality of vertical supports. The vertical support can be curved orbent to facilitate connection with the solid support. The verticalsupport can be arranged on the solid support to surround or outline theplurality of capture molecules immobilized on the solid support. Thevertical supports can be made of e.g., polypropylene, polystyrene,polyethylene, polymers, dextran, nylon, amylase, silicone, glass,natural and modified celluloses, polyacrylamides, agaroses, andmagnetite.

In one embodiment, the vertical support includes an adhesive. Theadhesive can be positioned on the solid support. After the verticalsupports are placed on the adhesive on the solid support anotheradhesive can be applied to the upper edges of the vertical supports. Thecover plate can then be placed upon the vertical supports. The verticalsupports inhibit the cover plate from contacting the solid support. Inthis manner, a channel can be formed between the solid support and thecover plate which allows fluid to pass into the channel.

The channel or channels formed by the solid support, vertical support,and cover plate can be round, trapezoidal, triangular or other geometricshapes as required. Channel sizes are optimally determined by theapplication. Channels can be from 0.01 mm to several millimeters deepand from 0.01 mm to several millimeters wide. Channels can be straight,curved, zig-zag, or U-shaped depending upon the application and specificfunction of the channel. Channels can be from 0.05 mm to severalmillimeters deep and from 0.1 to a centimeter or more in diameter.Capacity of the channels can range from nanoliters to 1 mL or moredepending upon the application. In another embodiment, the verticalsupports are SA2260, (Grace Biolabs); 1.5 mm (Wide)×1 mm (Thick)×65 mm(Long). In a related embodiment, the top adhesive layer of the SA2260 isremoved and the two sides are cut out from the product and usedseparately.

Absorbent Material

The device comprises an absorbent material at one end of the channel orchannels. The absorbent material comprises a material pervious to thepassage of fluid and is absorbent. Absorbent materials can include e.g.,plastics, polymers, acrylics, nylon, paper, cellulose, nitrocellulose,and ceramics. Other examples of absorbent materials include membranesavailable from the Pall Corporation (East Hills, N.Y.). The absorbentmaterial may or may not comprise pores. In one embodiment, the absorbentmaterial has pores. The pore sizes (cross-sectional dimension) of theabsorbent material can range between and including about 1 nanometer toabout 1 centimeter. Pore size can be adjusted according to theproperties of the sample and to control the rate of fluid movement orflow over the solid support. Preferably, the absorbent material providesfor wicking (i.e., drawing in of fluid by capillary action orcapillarity) of the fluid into the absorbent material. In order topromote wicking of the fluid into the absorbent material, the absorbentmaterial can also comprise a hydrophilic material, which can beprovided, for example, by the absorbent material itself with or withoutpost treating (e.g., plasma surface treatment such as hypercleaning,etching or micro-roughening, plasma surface modification of themolecular structure, surface chemical activation or crosslinking), or bya coating provided thereto, such as a surfactant. In another embodiment,the absorbent material used in a device of the invention results in alinear flow rate on the order of approximately 1 centimeter/minute.Additionally, flow rates can be adjusted (i.e., increased or decreased)by adding or subtracting material from the absorbent material.

In another embodiment, the absorbent material is Hi-Flow PlusNitrocellulose Membrane HF240 (Millipore; Billerica, Mass.).

Detection of Biomaker Bound to the Capture Molecules

In another embodiment, the device comprises a detection component fordetecting the biomarker bound to the sold support via the capturemolecules. The detection component can include a reader and a screen fordisplaying output from the reader. The reader can be optical. Oneembodiment of a detection component of the invention is the ScanArray™5000 XL (PerkinElmer, Inc.; Wellesley, Mass.). This is a benchtop,laser-based confocal scanning device with a photomultiplier tube (PMT)for sensitive fluorescence detection. Images collected onto a computercan be analyzed by QuantArray™ software. Raw intensities for each spotcan then be computed by taking the average of the logarithm of theintensity over all pixels in the region of interest that were greaterthan zero for quadruplicate spots on a slide and across duplicatechambers.

Another embodiment of a detection component of the invention is aimaging system that comprises a charge coupled device (CCD) camera. Thearrangement of this imaging system is shown in FIG. 2 and it consists ofa scientific-grade 16-bit, 1392×1040 pixel CCD camera (Lumenera Corp.MA), which is configured for Köhler epi-illumination of the samplemicroarray. The imaging system allows the sample to be illuminated fromthe front, while simultaneously being imaged from the same side by theCCD camera. Excitation light from a full-field White Lite® light 300 Wxenon arc lamp can be bandpass-filtered using a 525 nm excitation filter(Omega Optical Inc, Vt.) and focused uniformly on a sample using a setof two optic fiber cables (mellesgriot) held at an angle of 45 degrees.The spots can be focused onto the CCD using a camera lens (Infinimite®alpha, Edmund Optics) and filtered using a 600 nm longpass filter.Custom algorithms, built within the Lumenera camera software correct forCCD dark noise. Images saved in tiff format can be analyzed using theScanarray Express™ software (Perkin Elmer, Wellesley, Mass.). Outputfrom the imaging system can be displayed on a computer screen or otherviewing apparatus, including e.g., a liquid crystal display (LCD)device.

Arrays of the Invention

As describe herein, the device of the invention includes an arraycomprising a solid support, e.g., a glass slide and a plurality ofcapture molecules immobilized on the solid support. In one aspect, theinvention provides an array comprising a plurality of capture moleculesspecific for biomarkers associated with breast cancer. The biomarkerscan include, e.g., Her-2, MMP-2, CA 15-3, OPN, p53, VEGF, CA 125, andSerum Estrogen Receptor (SER). In one embodiment, the array includescapture molecules specific for CA-15-3 and OPN; in another embodimentthe array includes capture molecules specific for Her-2, MMP-2, CA 15-3,and OPN.

In a related aspect, the plurality of capture molecules are a pluralityof capture antibodies. The plurality of capture antibodies can includeat least two of the following capture antibodies: 1) anti-Her-2 (R&Dsystems; Monoclonal Anti-human ErbB2 Antibody; MAB-1129; Clone 191924),2) anti-MMP-2 (R&D systems; Monoclonal Anti-human MMP-2 Antibody;MAB-902; Clone 36006.211), 3) anti-CA 15-3 (Fitzgerald; MonoclonalAnti-human CA 15-3 Antibody; 10-C03; Clone M8071022), 4)anti-Osteopontin (R & D Systems; Monoclonal Anti-human OsteopontinAntibody; MAB-1433; Clone 190312), and 5) anti-VEGF (Biosource; VEGFpurified mouse anti-human; AHG011; Clone A183C-13G8). In one embodiment,the array includes 1) anti-Her-2 (R&D systems; Monoclonal Anti-humanErbB2 Antibody; MAB-1129; Clone 191924), 2) anti-MMP-2 (R&D systems;Monoclonal Anti-human MMP-2 Antibody; MAB-902; Clone 36006.211), 3)anti-CA 15-3 (Fitzgerald; Monoclonal Anti-human CA 15-3 Antibody;10-C03; Clone M8071022), 4) anti-Osteopontin (R & D Systems; MonoclonalAnti-human Osteopontin Antibody; MAB-1433; Clone 190312).

Methods for Categorizing a Sample

In one embodiment, the invention provides a method for scoring a samplefrom a subject, e.g., categorizing a human sample using quantitativedata associated with a plurality of biomarkers wherein the biomarkersare associated with breast cancer.

Samples

A sample can be derived from any subject of interest, includingmammalian subjects and, e.g., human subjects, e.g., patients. A samplecan include blood and other liquid samples of biological origin, solidtissue samples such as a biopsy specimen or tissue cultures or cellsderived therefrom, and the progeny thereof. A sample can be of cancerousorigin, e.g., breast cancer. A sample can comprise a single cell or morethan a single cell. Samples can also have been manipulated in any wayafter their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such as proteinsor polynucleotides. Sample also encompasses a clinical sample, and alsoincludes cells in culture, cell supernatants, and cell lysates.

Quantitative Data

A “dataset” of “quantitative data” is a set of numerical valuesresulting from evaluation of a sample (or population of samples) under adesired condition. In one embodiment, the quantitative data is proteinconcentration data. The values of the protein concentration data can beobtained, for example, by experimentally obtaining measures from asample and constructing a dataset from the measurements. In anotherembodiment, the protein concentration data is obtained using an enzymelinked immunosorbent assay (ELISA) format. In another embodiment, theprotein concentration data is obtained using a sandwich immunoassayformat. In a related embodiment, the protein concentration data isobtained using methods described herein, including sandwich immunoassayformats, and the following antibodies: Capture antibodies 1) anti-Her-2(R&D systems; Monoclonal Anti-human ErbB2 Antibody; MAB-1129; Clone191924), 2) anti-MMP-2 (R&D systems; Monoclonal Anti-human MMP-2Antibody; MAB-902; Clone 36006.211), 3) anti-CA 15-3 (Fitzgerald;Monoclonal Anti-human CA 15-3 Antibody; 10-C03; Clone M8071022), 4)anti-Osteopontin (R & D Systems; Monoclonal Anti-human OsteopontinAntibody; MAB-1433; Clone 190312), and 5) anti-VEGF (Biosource; VEGFpurified mouse anti-human; AHG011; Clone A183C-13G8); Detectionantibodies 1) anti-Her-2 (R & D Systems; Polyclonal Goat Anti-humanErbB2 Antibody; AF-1129), 2) anti-MMP-2 (R & D Systems; Polyclonal GoatAnti-human MMP-2 Antibody; AF-902), 3) anti-CA 15-3 (Fitzgerald;Monoclonal Anti-human CA 15-3 antibody; 10-C03B; Clone M8071021), 4)anti-Osteopontin (R & D Systems; Polyclonal Goat Anti-human OsteopontinAntibody; AF-1433), and 5) anti-VEGF (Biosource; Polyclonal RabbitAnti-human VEGF Biotin Conjugated Antibody; AHG9119). In anotherembodiment, the data are obtained using a multiplexed channel flow-baseddevice, e.g., the device described above (FIG. 1).

Alternatively, quantitative data can be obtained from a service providersuch as a laboratory, or from a database or a server on which the datahas been stored.

Plurality of Biomarkers

The method of categorizing sample uses data associated with a pluralityof biomarkers associated with breast cancer. In one aspect, theplurality of biomarkers associated with breast cancer includes CA 15-3and OPN. In another aspect, additional biomarkers can include Her-2. Inanother aspect, additional biomarkers can include MMP-2. In anotheraspect, additional biomarkers can include VEGF. In another aspect,additional biomarkers can include Her-2 and MMP-2. In yet anotheraspect, additional biomarkers can further include p53, CA 125, and SerumEstrogen Receptor (SER). In one embodiment of the invention, the methodof categorizing a sample uses data associated with the followingbiomarkers: CA 15-3, OPN, Her-2, and MMP-2.

Scoring the Sample

In one embodiment, scoring the sample comprises analyzing the data andoutputting a score. Analysis of the data can include use of a predictivemodel. Predictive models can be developed using, e.g., principalcomponent analysis (PCA), and linear discriminant analysis.

PCA is a technique used to reduce multidimensional data sets to lowerdimensions for analysis. Mathematically, PCA is defined as an orthogonallinear transformation that transforms the data to a new coordinatesystem such that the greatest variance by any projection of the datacomes to lie on the first coordinate (called the first principalcomponent), the second greatest variance on the second coordinate, andso on. PCA can be used as a tool in exploratory data analysis and formaking predictive models. PCA can also involve the calculation of theeigenvalue decomposition of a data covariance matrix or singular valuedecomposition of a data matrix, usually after mean centering the datafor each attribute. The results of a PCA are usually discussed in termsof component scores and loadings.

Linear discriminant analysis is a method used to find the linearcombination of features which best separate two or more classes ofobjects or events. The resulting combination can be used as a linearclassifier, or, alternatively, for dimensionality reduction before laterclassification.

In another embodiment, the analysis can include categorizing the sampleaccording to a predictive model. The probability that categorization iscorrect is model- and biomarker-dependent and can be at least 60%, atleast 70%, at least 80%, at least 87%, at least 90%, or at least 95%correct. Categories can include a healthy categorization, i.e.disease-free, an early-stage disease categorization, and a late-stagedisease categorization.

In another embodiment, the score can be compared to a second scoredetermined for a second sample from the mammalian subject. Thiscomparison can be used e.g., to determine the progress of therapy forthe treatment of disease. In yet another embodiment, the score can beused to diagnose a neoplastic breast disease. A neoplastic breastdisease can include e.g., breast cancer.

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W.H.Freeman and Company, 1993); A. L. Lehninger, Biochemistry (WorthPublishers, Inc., current addition); Sambrook, et al., MolecularCloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology(S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Carey and Sundberg Advanced Organic Chemistry 3^(rd) Ed.(Plenum Press) Vols A and B (1992).

Example 1 Development of Multiplexed Assays

A multiplexed immunoassay for the measurement of breast cancerbiomarkers using the protein microarray format was developed.

Protein microarrays have the potential to be used to simultaneouslyanalyze large numbers of serum proteins in a rapid and reproduciblemanner. In recent years, protein microarrays have evolved as powerfultools to address high-throughput requirements by performing“traditional” biochemistry in an ultra-high-throughput and miniaturizedformat. On protein microarrays, purified capture molecules areimmobilized in unique locations on the surface of the substrate thatallow for recognizing the target under study. Since each capturemolecule is immobilized in a precise, predetermined spot, the proteinmicroarrays achieve multiplexing capability based on this uniquelocation of the capture molecule and therefore the target protein boundto it (assuming high specificity of the antibody-antigen reaction). Inthe case of a reverse assay on the microarray, the immobilized moleculeincludes large collections of purified proteins or samples such as seraand lysates, which is then probed with a single antibody.

Materials

A multiplexed breast cancer assay based on the protein microarray wasdeveloped after validating the reagents using 2d Gel Electrophoresis andELISA techniques. This example presents the assay development materialsand methods on these platforms.

Common Reagents

Recombinant proteins, capture, and biotinylated detection antibodies forHer-2, MMP-2 and Osteopontin were purchased from R&D systems(Minneapolis, Minn.). Other reagents used in the assay include: VEGFantigen and capture and biotinylated detection antibodies (BiosourceInternational Camarillo, Calif.), CA 15-3 antigen and anti-CA 15-3capture and detection antibodies (Fitzgerald, Concord, Mass.). The CA15-3 detection antibody was biotinylated using a kit and according tothe manufacturer's (Pierce, Rockford, Ill.) instructions. All otherdetection antibodies were purchased as biotin conjugates. Lyophilizedhuman serum was purchased from Rockland Immunochemicals (Gilbertsville,Pa.). Capture antibodies used were: 1) Her-2 (R&D systems; MonoclonalAnti-human ErbB2 Antibody; MAB-1129; Clone 191924), 2) MMP-2 (R&Dsystems; Monoclonal Anti-human MMP-2 Antibody; MAB-902; Clone36006.211), 3) CA 15-3 (Fitzgerald; Monoclonal Anti-human CA 15-3Antibody; 10-C03; Clone M8071022), 4) Osteopontin (R & D Systems;Monoclonal Anti-human Osteopontin Antibody; MAB-1433; Clone 190312), and5) VEGF (Biosource; VEGF purified mouse anti-human; AHG011; CloneA183C-13G8). Detection antibodies used were: 1) Her-2 (R & D Systems;Polyclonal Goat Anti-human ErbB2 Antibody; AF-1129), 2) MMP-2 (R & DSystems; Polyclonal Goat Anti-human MMP-2 Antibody; AF-902), 3) CA 15-3(Fitzgerald; Monoclonal Anti-human CA 15-3 antibody; 10-C03B; CloneM8071021), 4) Osteopontin (R & D Systems; Polyclonal Goat Anti-humanOsteopontin Antibody; AF-1433), and 5) VEGF (Biosource; PolyclonalRabbit Anti-human VEGF Biotin Conjugated Antibody; AHG9119).

Reagents for Western Blotting

Anti-goat, anti-rabbit and anti-mouse secondary antibodies wereconjugated to horseradish peroxidase (HRP) (EMD Biosciences, San Diego,Calif.). Laemmli sample buffer, precision plus dual molecular weightstandard and 7.5% and 10% ready gels were purchased from BioRad Inc(Hercules, Calif.), enhanced chemiluminescence (ECL) detection kits andhyperfilm were obtained from Amersham Biosciences (Piscataway, N.J.) andPVDF transfer membrane from (Millipore, Bedford, Mass.). Tris bufferedsaline (TBS) and Tris buffered saline with 0.05% tween (TBS-T), waspurchased from Sigma-Aldrich (St. Louis, Mo.).

Reagents for ELISA

Antibody biotinylation kit and Blotto were purchased from Pierce(Rockford, Ill.). Vectastain kit was purchased from Vector Labs(Burlingame, Calif.), Nunc ELISA 96 well plates was obtained from NalgeNunc International, (Rochester, N.Y.) and Hanks buffer was purchasedfrom Invitrogen—GIBCO (Carlsbad, Calif.). Phosphate buffered saline with0.05% tween (PBS-T), Phosphate buffered saline (PBS) and TMB(3,3′.5,5′-tetramethylbenzidine) were purchased from Sigma-Aldrich (St.Louis, Mo.).

Reagents for Protein Microarrays

Streptavidin conjugated Alexa 546 was purchased fromInvitrogen—Molecular Probes (Carlsbad, Calif.), GAPS II™ slides werepurchased from Corning LifeSciences (Corning, N.Y.) and BSA waspurchased from Sigma-Aldrich (St. Louis, Mo.).

Methods

Reagent Quality Validation Using Gel Electrophoresis and WesternBlotting

Gel electrophoresis for each biomarker was performed in non-reducingconditions with 7.5% (for Her-2 (185 kD) and CA 15-3 (250 kD)) and 10%(for OPN (66 kD), MMP-2 (72 kD) and VEGF (38 kD)) polyacrylamide gels.Samples were solubilized in Laemmli sample buffer and boiled for 3minutes before loading into the sample wells. Approximately 150 μg ofeach protein was loaded into each well. Precision plus dual standard wasused as the molecular weight marker. After electrophoresis for 2 h at120V, the proteins were transferred from the gel on to the PVDF membraneunder an electric field, using a fully immersed wet unit (BioRad,Hercules, Calif.), for 1 h at room temperature with ice in the unit. Themembrane was then immersed in wash buffer (TBS-T) for 15 minutes andblocked overnight at 4° C., with 5% w/v milk (BioRad, Hercules, Calif.)prepared in wash buffer. Following the blocking step, the membrane waswashed in TBS-T for 30 minutes and incubated with primary antibody at500 ng/ml (Mouse and Goat anti Her-2 antibody, Mouse and Goat anti MMP-2antibody, Mouse and Goat anti-Osteopontin antibody, Mouse and Rabbitanti-VEGF antibody and two Mouse anti-CA 15-3 antibodies) for 1 hour atroom temperature. After a 30 minute wash in TBS-T, the membrane wasincubated for 1 hour at room temperature with the secondary antibodyconjugated to HRP (Anti-Goat, Anti-Rabbit or Anti-Mouse as applicable)at 500 ng/ml. The protein bands in the gel were finally visualized usingthe ECL kit and a sensitive photo film.

Selection of Antibody Pairs: Enyzme Linked Immunosorbent Assays (ELISA)

The concentrations of the capture and detector antibodies for all fivebiomarkers, to be used in a sandwich format, were optimized usingstandard two antibody sandwich ELISA. 100 μL of capture antibodysolution in ELISA coating buffer (Hanks buffer, with 0.375% NaHCO₃) wasadded in duplicate to the wells of the 96-well plate in a series of fourdilutions (0.1 μg/ml, 0.3 μg/ml, 1 μg/ml and 3 μg/ml). These dilutionswere added across the rows of the plate. After overnight incubation at4° C., the coating antibody solution was aspirated from the wells andthe plates were rinsed 6 times in PBS-T and then blocked for 2 h at roomtemperature in 200 μl of BLOTTO. The solution was then aspirated fromthe wells, and the plate was washed 6 times in PBS-T. 100 μL of therecombinant antigen was added to the appropriate wells in dilutionsrepresenting the middle portion of the clinical range for thebiomarkers. The concentrations of antigens used for this assay were 10ng/ml for Her-2, 600 ng/ml for MMP-2, 130 U/ml for CA 15-3, 700 ng/mlfor OPN, and 550 pg/ml for VEGF, respectively. Buffer without antigenwas used to represent background signal. The plate was sealed andincubated at room temperature for 2 hours followed by 6 washes withPBS-T. 100 μl of biotinylated detection antibody solutions were thenadded down the columns of the plate in a series of four dilutions (0.36μg/ml, 1.1 μg/ml, 3.3 μg/ml and 9.9 μg/ml). For VEGF, biotinylatedantibody was used at higher concentrations of 3 μg/ml, 8 μg/ml, 25 μg/mland 75 μg/ml according to manufacturer's (R&D systems) suggestions.Following a 1-hour incubation at room temperature, the plates werewashed 6 times and 100 μl of Vectastain solution (prepared according tothe manufacturer's instructions) was added for 30 minutes to probe forthe detection antibodies. The plates were then washed 6 times andincubated with 100 μl of TMB for 30 minutes at room temperature. Thereaction was stopped by adding 50L of 1N H₂SO₄ to each well beforereading the absorbance at 485 nm in the microplate reader (BioRad,Hercules, Calif.). After color development and measurement ofabsorbance, the capture and detector antibody concentrations yieldingthe best signal to noise ratio were selected for further ELISAdevelopment.

Standard curves of each of the biomarkers were then obtained byperforming the ELISA as outlined in the basic protocol above except thatoptimized concentrations of capture and detector antibodies were usedwith 8 serial dilutions of recombinant antigen standards to obtain astandard titration curve. The concentrations of biomarkers used toestablish this standard curve were 1 ng/ml-128 ng/ml for Her-2, 10ng/ml-1280 ng/ml for MMP-2, 2 U/ml-256 U/ml for CA 15-3, 10 ng/ml-1280ng/ml for OPN and 1 pg/ml-1280 pg/ml for VEGF.

Microarray Spotting and Assay Protocol

Concentrations of capture and detector antibody pair for each of thefive biomarkers were optimized for use in a sandwich assay format on themicroarray similar to the ELISA optimizations. Aminosilanated, GAPS II™barcoded glass slides were spotted with optimized dilutions of captureantibodies using a robotic arrayer (Norgen Systems Inc.; Mountain View,Calif.). Four print heads were used to deposit approximately 1 nl ofcapture antibody solution, generating a total of 8 arrays per slide with250 μm diameter spots with a spot-to-spot distance of 350 μm. The layoutof each 8×12 array of printed antibody spots corresponded to one spotper well in a standard 8×12 (96-well) format. These capture antibodieswere printed in a series of four dilutions (1000 μg/ml, 500 μg/ml, 250μg/ml and 125 μg/ml). Also printed on each slide were two controls.Bovine serum albumin (BSA) served as negative control (NC). Alexa 546spots were used as position controls (PC), which served as referencepoints when the slides were imaged. The spotted slides were cross-linkedunder ultraviolet light for 5 minutes and were stored in the dark at 4°C.

The eight arrays were separated using silicone gasket chambers(Schleicher & Schuell Bioscience, Keene N.H.) and were blocked with 1mg/ml BSA solution for 30 minutes. The protein microarrays were thenwashed for 15 minutes and incubated with 100 μl of target antigen atdilutions representing the middle portion of the clinically significantranges for the biomarkers. The concentrations of antigens used for thisassay were 10 ng/ml for Her-2, 600 ng/ml for MMP-2, 130 U/ml for CA15-3, 700 ng/ml for OPN and 550 pg/ml for VEGF respectively. Bufferwithout any added recombinant antigen was used to determine thebackground due to non-specific binding on the arrays. The solution wasaspirated and washed with PBS-T for 15 minutes and the wells were thenincubated with 100 μl of a series of four dilutions of biotinylatedantibody solution (1.8 μg/ml, 3.75 μg/ml, 7.5 μg/ml and 15 μg/ml) in PBSfor 30 minutes. Following a 15 minute wash, the arrays were incubatedwith 100 μl streptavidin conjugated Alexa 546 for 10 minutes. Thechambers were then removed and the slides were agitated in PBS-T for 10minutes and dried by centrifugation prior to scanning.

Two sets of standard curves were obtained. One, using PBS as thediluting medium for the recombinant antigens and one using human serumas the medium. The protein microarray standard titration curves wereobtained as outlined in the basic protocol except that serial dilutionsof recombinant antigen standards in PBS and human serum were used asanalytes and the optimized concentrations of capture and detectorantibodies were used for detection. The concentrations of biomarkersused to establish this standard curve were 1 ng/ml-128 ng/ml for Her-2,10 ng/ml-1280 ng/ml for MMP-2, 2 U/ml-256 U/ml for CA 15-3, 10ng/ml-1280 ng/ml for OPN, and 1 pg/ml-1280 pg/ml for VEGF.

Microarray Spotting and Assay Protocol: Multiplex Curves

This multiplexed assay was performed essentially as described for thestandard titration curves. Capture antibodies for Her-2, MMP-2, OPN, CA15-3 and VEGF were spotted in quadruplicate at 500 μg/ml on the GAPS II™slides to form a 4×5 array grid. Ten different multiplex samples wereprepared. Five samples were prepared with a mixture of all but oneantigen and five of the remaining samples contained only one antigeneach. The concentrations of recombinant antigens used in this assay were20 ng/ml Her-2, 800 ng/ml MMP-2, 130 U/ml CA 15-3, 900 ng/ml OPN and 950pg/ml VEGF. A second set often slides was prepared in a similar manner,but using human serum as the medium instead of PBS. In this case, onlyfour biomarkers were used (Her-2, MMP-2, CA 15-3 and Osteopontin).Antibody microarrays were incubated with these antigen samples induplicate, followed by incubation with a detector antibody “cocktail”containing biotinylated antibodies for all five biomarkers at aconcentration of 3.75 μg/ml. Streptavidin conjugated Alexa 546 was usedas the reporter at a concentration of 5 μg/ml. The chambers were thenremoved and the slides were agitated in PBS-T for 10 minutes and driedby centrifugation prior to scanning.

Microarray Imaging and Analysis Protocol

Slides were imaged with ScanArray 5000 XL (Perkin Elmer, Wellesley,Mass.), which is a laser-based confocal scanner, at 543 nm excitation.Images collected onto a PC were analyzed by Scanarray Express™ software(Perkin Elmer, Wellesley, Mass.). Raw intensities for each spot werecomputed by taking the average of the logarithm of the intensity overall pixels in the region of interest that were greater than zero for thespot. A median of all quadruplicate spots across 2 wells (resulting in atotal of eight spots per sample) was computed and plotted againstconcentration for a titration curve.

Results

Western Blotting

Antigens purchased from various sources were tested for purity byperforming SDS-PAGE, which demonstrated the specificities of theantibodies as well as tested the purity of the recombinant antigens.This was followed by detection with monoclonal antibodies (FIG. 3A) (tobe employed as capture antibodies in the ELISA) polyclonal antibodies(FIG. 3B) (to be employed as detector antibodies in the ELISA) usingWestern Blotting. Approximately 150 ng of protein was loaded in eachlane and the gels were run under non-reducing conditions to mimic thedetection of proteins in serum. Distinct bands are observed for four ofthe biomarkers, MMP-2 (72 kDa), CA 15-3 (250 kDa), OPN (66 kDa) and VEGF(38 kDa). For the biomarker, Her-2 (185 kDa) distinct, multiple bandswere seen due to the phosphorylated forms of the protein, all of whichare recognized by the antibody.

To demonstrate the specificity of these antibodies, all five recombinantprotein biomarkers were run in neighboring wells in the gel, transferredthem onto the PVDF membrane and incubated the membrane with one singleantibody. As can be seen in FIG. 3, the capture (A-E) and detector (F-J)antibodies showed high specificity to their respective antigens. Usingthis method, the most specific capture (monoclonal) antibodies anddetector (monoclonal and polyclonal) for all five biomarkers from atotal of 30 antibody pairs and 5 recombinant antigens were selected.

ELISA

The selected antibodies were then characterized for dynamic range andsensitivity in the clinically-relevant concentrations (as observed innormal and breast cancer patient sera) using ELISA as the validationmethod. The monoclonal antibodies were used as capture and thebiotinylated polyclonal antibodies were used as detector (except in thecase of CA 15-3, where both antibodies were monoclonal). ELISA plateswere coated with four different concentrations of the capture antibodiesin the ELISA coating buffer as described in materials and methods. Twodilutions of recombinant antigen were used, one on either end of theclinically-relevant range. Sample with no antigen added was used asnegative control for the assay. Biotinylated detector antibodies werethen added in four different dilutions to the wells. FIG. 4 shows theresults obtained from these optimizations. The concentrations of captureand detector antibodies that yielded a good signal (O. D between 1.0 and2.0, which was neither too low nor saturated) were chosen for theassays. Since the detector antibodies were more expensive than thecapture antibodies (because of their biotin-conjugation), a combinationof capture and detection concentration was chosen that gave a goodsignal, but nevertheless used the minimum amount of detector antibody.Table 1 lists the final chosen concentrations of both the antibodiesusing this criterion and which were used to develop ELISA curves.

Standard curves for all five biomarkers were developed on the ELISAplatform. 96 well polystyrene plates were coated with capture antibodyat optimized concentration, followed by incubation with 8 serialdilutions of recombinant antigen in PBS. The concentrations ofbiomarkers used to establish this standard curves were 1 ng/ml-128 ng/mlfor Her-2, 10 ng/ml-1280 ng/ml for MMP-2, 2 U/ml-256 U/ml for CA 15-3,10 ng/ml-1280 ng/ml for OPN and 1 pg/ml-1280 pg/ml for VEGF. The wellswere then probed with optimized concentration of biotinylated detectorantibody. Enzyme based detection was used in this assay in which, wellswere interrogated with a spectrophotometer to obtain intensities ofsubstrate color. Increased color intensity was observed with increasedprotein concentration. These intensities are quantified and plotted as afunction of antigen concentration to obtain a standard curve. FIG. 5demonstrates that the ELISA assay curves are linear over theclinically-relevant ranges for these biomarkers and span normal as wellas elevated levels as seen in cancer. Data points for each curverepresent the average intensities of two replicate samples.Reproducibility was determined from the coefficient of variation, whichwas approximately 5% for all protein biomarker curves. The backgroundsignal, which is a measure of non-specific binding, was considered to bethe signal from the wells in which no antigen was added. Each point onthe ELISA curve plotted below represents the signal from the wells minusthis background.

Standard Curves were Established on Protein Microarrays

Western blotting was used to validate the specificity of the reagentsselected and ELISA was used to characterize the performance of theseantibodies in a sandwich immunoassay format. These antibody pairs werethen employed on the protein microarray platform to generate standardcurves in singleplex as well as multiplex format. Similar to the ELISA,concentrations of these antibodies were optimized on the proteinmicroarray prior to establishing the standard curves. A schematicdiagram of the microarray assay approach used in this study is shown inFIG. 6.

Capture antibodies immobilized on modified glass slides are probed withsample containing antigen. A second biotinylated antibody then binds tothe antigen on the array and a streptavidin-linked fluorescent dye wasused for detection. For the optimization studies, amine modifiedmicroarray slides were printed with four distinct dilutions of thecapture antibodies in PBS buffer as described in materials and methods.The spot size was approximately 250 μm in diameter. Two dilutions ofrecombinant antigen were used, one on either end of theclinically-relevant range. Sample with no antigen added was used asnegative control for the assay. A second, biotinylated antibodyrecognizing a different epitope on the same antigen was used in threedifferent dilutions for detection. This “sandwich” approach favorsspecificity in analyte detection, since the two separate antibodiessequentially enable selective detection. A streptavidin-Alexa 546fluorescent reporter was then used to bind to the biotin moiety of thedetection antibody which then produced fluorescent signals proportionalto the amount of antigen bound on the array.

These fluorescent spots were then quantified using a fluorescencemicroarray reader. FIG. 7 illustrates the signals obtained from theseoptimizations and Table 2 demonstrates the final chosen concentrationsof both the antibodies that were used to develop microarray curves.Similar to the ELISA optimization experiments, the combination ofcapture and detector antibody that yielded maximum response with minimumamount of detector antibody was chosen as the optimized concentration.

The optimized concentrations of capture antibodies were printed onmodified microarray slides using a robotic arrayer such that eachantibody was present in quadruplicate. The capabilities of themicroarray were further tested by analyzing single biomarkers over arange of concentrations in a multiplexed format, using all five captureand detector antibodies (see FIGS. 8 and 9). The goal here was tomeasure the effect of the presence of other capture agents and varyingantibody affinities on the multiplexed detection of five proteins.Protein microarrays were incubated with 8 serial dilutions ofrecombinant antigen diluted in PBS. The concentration ranges used were 1ng/ml-128 ng/ml for Her-2, 10 ng/ml-1280 ng/ml for MMP-2, 2 U/ml-256U/ml for CA 15-3, 10 ng/ml-1280 ng/ml for OPN and 10 pg/ml-1280 pg/mlfor VEGF. Standard curves were obtained on the protein microarray formatfor each biomarker in four formats. First, only one capture antibody(Singleplex format) was used and second, the all five capture antibodieswere spotted on the array and all five detector antibodies were used(Multiplex format). In both cases the antigens were diluted in PBS. Thismethod was most similar to the ELISA. The third and fourth formats aresimilar to the first and second, except that human serum was used as thediluting medium instead of PBS. This method enabled the quantificationof effect of serum on background noise and thus simulated detection ofthese proteins in patient sera.

FIG. 8 shows a composite image of eight different arrays with eachcolumn representing a different array that was incubated with increasingantigen concentration from right side of the FIG. to the left side ofthe FIG. Slides shown in Panel A were incubated with Her-2 atconcentrations ranging from 0 ng/ml (right) to 128 ng/ml (left). Slidesshown in Panel B were incubated with MMP-2 with a concentration range of0 ng/ml (right) to 1280 ng/ml (left). Slides shown in Panel C wereincubated with CA 15-3 at concentrations ranging from 0 U/ml (right)-256U/ml (left) and those shown in Panel D were incubated with Osteopontinat concentrations from 0 ng/ml (right) to 1280 ng/ml (left). Finally,Panel E shows slides incubated with VEGF in the concentration range of 0pg/ml (right)-1280 pg/ml (left). The results show increased fluorescenceintensity with increased protein concentration.

The fluorescence from these spots is quantified using the Scanarraysoftware and plotted as a function of antigen concentration in FIG. 9(A-E) for the experiments with PBS as the medium and in FIG. 9(F-J) forthe experiments in which human serum was used as the diluent. Thebackground signal, which is a measure of non-specific binding, wasconsidered to be the signal from the arrays in which no antigen wasadded. Data points for each curve represent the average intensities ofeight replicates (background subtracted) obtained using quadruplicatespots in two replicate arrays. Reproducibility was determined by thecoefficient of variation, which was approximately 15% for all proteinbiomarker curves.

The multiplex assays have a higher background noise level than thesingleplex assays probably due to the presence of additional detectorantibodies in the assay. However, the shapes of the intensity curvescompare well to the assays in the singleplex format validating the useof multiplex microarray assays. A higher background in the serum assaysas compared to the PBS based assays was observed, likely due to thepresence of other proteins in the serum that bind non-specifically tothe slide.

Comparison of ELISA and Protein Microarray Formats

The developed microarray immunoassay offers many advantages over thetraditional ELISA technique, including smaller size, lower costs, andmultiplexing capability. Similar to the ELISA, we carefully optimizedthe concentrations of antibodies on the microarray to yield sensitivedose response curves. FIG. 10 shows the comparison of the standardcurves obtained for each of the biomarkers using microarray technologyand the ELISA in a scatter plot. Protein microarray shows similardynamic range. Data from the two methods showed a linear relationshipwith a correlation coefficient (r²) of greater then 0.97, indicatingthat both methods produce similar results.

The background noise due to non-specific adsorption of protein on themicroarray glass surface increases with increasing proteinconcentration. Therefore, the detection of a specific target protein islimited not only by its concentration, but also by the concentration ofthe other proteins in the mixture. Serum is a complex mixture of manyproteins with a very high concentration (˜85 mg/ml) compared to PBS (0mg/ml) causing significantly higher background levels in the assaysdeveloped using serum instead of PBS as the medium. The four biomarkerswere detectable above this noise level in this assay. However, forproteins present in very small amounts (pM or fM) in serum, the highbackground can reduce the sensitivity of the assay. In that case, theabundant proteins (such as albumin) could be filtered out and the serumcould be concentrated to enhance the detection limit. The signal can beamplified using specific secondary antibody molecules.

Microarray Multiplexed Assays

To demonstrate that this array technology could be used tosimultaneously detect multiple biomarkers, all five protein biomarkerswere analyzed in one single microarray. In this experiment, twelveidentical slides were printed with capture antibodies to the fiveprotein biomarkers. One of these slides was incubated with a mixture ofall five antigens in the high concentrations observed in cancer, whileone slide was incubated with a mixture of all five antigens in the lowerconcentrations observed in normal sera. Five slides were incubated withthe same mixture of proteins containing all but one biomarker and theother five slides were incubated with only one antigen. The specificityof our immunoassays is illustrated in the FIG. 11(A).

Four (Her-2, MMP-2, CA 15-3 and OPN) of the five biomarkers diluted inPBS were able to be detected, simultaneously with high sensitivity andspecificity in the assays. However no signal was observed for VEGF evenat high concentrations. While VEGF could be sensitively detected in asingleplex assay (FIGS. 8E and 9E) very strong laser settings(power=100% and PMT gain 95%) had to be used. This posed a problem inthe multiplexed format where the other biomarkers were sensitivelydetected at lower laser settings (70% laser power and 75% PMT gain).Therefore, two scans were performed, one at the lower setting to obtainfluorescence signals from the four biomarkers and one at the highersetting to obtain fluorescent signals from VEGF. No VEGF signal could beobserved at the low laser setting and at the higher laser setting,tremendously high backgrounds were observed with reduced the signal tonoise ratios for all the biomarkers. Therefore, in a further effort toselectively increase signal strength from VEGF, a biotinylated goatanti-rabbit secondary antibody specific to the biotinylated rabbitanti-VEGF antibody in the mixture was used. In this procedure, themicroarray was first incubated with the antigen mixture followed byincubation with biotinylated antibodies to the five biomarkers, Her-2,CA 15-3, MMP-2, OPN and VEGF. The microarray was subsequently incubatedwith biotin-anti rabbit secondary antibody. This step was introduced tospecifically amplify the VEGF signal since the biotinylated antibodiesfor the other 4 biomarkers were prepared in goat or mouse as the host.However, this antibody cross-reacted with the monoclonal captureantibodies for all the biomarkers, thus generating very high backgroundnoise. Recombinant antibodies engineered for affinity and specificitycan in the future improve the multiplexing capability of assays byeliminating cross-reactivity and by increasing sensitivity. This wouldoffer the antibody arrays a flexible, quantitative range and therebyincreasing the pool of biomarkers that can be potentially assayedsimultaneously.

A multiplexed assay similar to the one described above was developed,but replacing PBS with human serum as the medium. The results from thisassay are shown in FIG. 11 (B). In assays where only one antigen waspreset it was observed that the signal from the other spots is notnegative. This is also observed when a particular antigen is left out ofthe mixture, as serum contains these proteins in low concentrationsunlike PBS, which contains no protein at all. Also the overallbackground noise arising from serum on the slide is much higher than thePBS arrays, similar to the effect observed in the standard curves usingserum.

Example 2 Sensitive Multiplexed Diagnostic Test for Breast Disease

The multiplexed protein microarray assay developed previously is appliedto the differential detection of four biomarkers in breast cancerpatient sera.

Materials and Methods

Microarray assay reagents and protocols are essentially similar to thosedescribed above in Example 1. Recombinant proteins, capture andbiotinylated detection antibodies for Her-2, MMP-2 and Osteopontin werepurchased from R&D systems (Minneapolis, Minn.). Other reagents used inthe assay include: VEGF antigen and capture and biotinylated detectionantibodies (Biosource International Camarillo, Calif.), CA 15-3 antigenand anti-CA 15-3 capture and detection antibodies (Fitzgerald, Concord,Mass.). The CA 15-3 detection antibody was biotinylated using a kit andaccording to the manufacturer's (Pierce, Rockford, Ill.) instructions.All other detection antibodies were purchased as biotin conjugates.Lyophilized human serum was purchased from Rockland Immunochemicals(Gilbertsville, Pa.). Streptavidin conjugated Alexa 546 was purchasedfrom Invitrogen—Molecular Probes (Carlsbad, Calif.), GAPS II™ slideswere purchased from Corning LifeSciences (Corning, N.Y.) and BSA waspurchased from Sigma-Aldrich (St. Louis, Mo.). Sera from 41 metastaticbreast cancer patients, 33 breast cancer patients with early stagedisease and 39 controls were obtained from the Breast Cancer SerumResource, Lombardi Cancer Center (Washington, D.C.). Capture antibodiesused were: 1) Her-2 (R&D systems; Monoclonal Anti-human ErbB2 Antibody;MAB-1129; Clone 191924), 2) MMP-2 (R&D systems; Monoclonal Anti-humanMMP-2 Antibody; MAB-902; Clone 36006.211), 3) CA 15-3 (Fitzgerald;Monoclonal Anti-human CA 15-3 Antibody; 10-C03; Clone M8071022), 4)Osteopontin (R & D Systems; Monoclonal Anti-human Osteopontin Antibody;MAB-1433; Clone 190312), and 5) VEGF (Biosource; VEGF purified mouseanti-human; AHG011; Clone A183C-13G8). Detection antibodies usedwere: 1) Her-2 (R & D Systems; Polyclonal Goat Anti-human ErbB2Antibody; AF-1129), 2) MMP-2 (R & D Systems; Polyclonal Goat Anti-humanMMP-2 Antibody; AF-902), 3) CA 15-3 (Fitzgerald; Monoclonal Anti-humanCA 15-3 antibody; 10-C03B; Clone M8071021), 4) Osteopontin (R & DSystems; Polyclonal Goat Anti-human Osteopontin Antibody; AF-1433), and5) VEGF (Biosource; Polyclonal Rabbit Anti-human VEGF Biotin ConjugatedAntibody; AHG9119).

Microarray Spotting Protocol

Aminosilanated, GAPS II™ barcoded glass slides were spotted with captureantibodies using a robotic arrayer (Norgen Systems Inc.; Mountain View,Calif.) in quadruplicate at 500 μg/ml to form a 4×4 array grid. Fourprint heads were used to deposit approximately 1 nl of capture antibodysolution, generating a total of 8 arrays per slide with 250 μm diameterspots and with a spot-to-spot distance of 350 μm. The spotted slideswere cross-linked under ultraviolet light for 5 minutes and were storedin the dark at 4° C.

Multiplexed Microarray Assay Protocol with Patient Sera: Pilot Study

The eight arrays were separated from each other using silicone gasketchambers (Schleicher & Schuell Bioscience, Keene N.H.) and were blockedwith 1 mg/ml BSA solution for 30 minutes followed by incubation with 50μl of patient serum sample for 60 min. A total of 30 different patientserum samples were used in this study (10 metastatic, 10 early stage and10 control). This experiment was performed in duplicate, for control andearly stage patients, but replicates were not used for metastaticpatients due to limited availability of samples. Arrays were then washedin PBS-T for 15 min followed by incubation with detector antibody“cocktail” containing biotinylated antibodies for all four biomarkers ata concentration of 3.75 μg/ml for 60 min. Streptavidin conjugated Alexa546 was used as the reporter at a concentration of 5 μg/ml for 10 min.The chambers were then removed and the slides were agitated in PBS-T for10 minutes and dried by centrifugation prior to scanning.

Multiplexed Microarray Assay Protocol with Patient Sera: Larger SampleSet

Multiplexed capture antibody arrays were printed as described in theabove section. In this assay, two control molecules were also includedon the slide. Bovine serum albumin (BSA) served as negative control(NC). Alexa 546 spots were used as positive controls (PC). A total of 87patient samples were used in this study, 29 of which were metastatic, 29were early stage and the other 29 were control samples. The experimentwas also planned such that each slide contained all the three samplecategories (Control, Early stage and Metastatic) and each sample wassplit into two aliquots, which were placed on different slides. Thisarrangement accounted for inter-slide technical variation. Themicroarray assay was performed in a manner similar to Example 1.

Microarray Assay Protocol: Standard Curves

In this case, the protein microarrays were incubated with 50 μl ofrecombinant antigen diluted in human serum for 60 min, after theblocking step with BSA. The concentrations of biomarkers used toestablish these standard curves were 1 ng/ml-128 ng/ml for Her-2, 10ng/ml-1280 ng/ml for MMP-2, 2 U/ml-256 U/ml for CA 15-3, 10 ng/ml-1280ng/ml for OPN, and 1 pg/ml-1280 pg/ml for VEGF. Serum without any addedrecombinant antigen was used to determine the baseline levels of thebiomarkers. The solution was aspirated and washed with PBS-T for 15minutes and the wells were then incubated with 100 μl of biotinylatedantibody solution for 60 minutes. Following a 15 minute wash, the arrayswere incubated with 100 μl streptavidin conjugated Alexa 546 for 10minutes. The chambers were then removed and the slides were agitated inPBS-T for 10 minutes and dried by centrifugation prior to scanning.

Microarray Imaging and Analysis Protocol

Slides were imaged with ScanArray 5000 XL (Perkin Elmer, Wellesley,Mass.), which is a laser-based confocal scanner, at 543 nm excitation.Images collected onto a PC were analyzed by Scanarray Express™ software(Perkin Elmer, Wellesley, Mass.). Raw intensities for each spot werecomputed by taking the average of the logarithm of the intensity overall pixels in the region of interest that were greater than zero for thespot. Median fluorescent intensities of all four spots for eachbiomarker were then computed for all patient samples. Principalcomponent analysis was used to linearize the four dimensional dataobtained from the measurement of four biomarker levels. Lineardiscriminant analysis was used to measure the accuracy of classificationof unknown samples into appropriate disease categories (control, earlystage or metastatic) based on biomarker levels. Both of thesestatistical functions were carried out using MATLAB.

Results

Antibody arrays were prepared by spotting multiple capture antibodies onamine-modified glass slides. These arrays were incubated with 50 μlbreast cancer patient serum samples that were arranged on the slides asshown in FIG. 12. The notation C stands for control populations, E forearly stage patients and M for patients with metastatic disease. A totalof 30 patient sera were used; 10 controls, 10 early stage and 10metastatic. The control and early stage samples were used in duplicatealiquots and the metastatic samples were used in single aliquots due tolimited amount of sample.

FIG. 13 shows the quantification of the fluorescent intensities usingbox plots, which is an efficient method for displaying a five-pointsummary of the data. Median fluorescent signals were obtained for eachof the biomarkers and their log (to the base 2) values were plotted inthe FIG. for each disease state. This demonstrated the effect of diseasestate on individual marker concentrations in serum. The upper boundaryor hinge of the box represents the 75^(th) percentile of the data andthe lower boundary represents the 25^(th) percentile of the data. Thus,the box represents the middle 50% of the data and this region is calledthe inter-quartile range. The dot inside the box represents the medianvalue of the data. In this case, the data is skewed since the medianvalue is not equidistant from the hinges. The ends of the vertical linesor whiskers indicate 1.5 times the value of the inter-quartile range,while the spots outside the whiskers denote the outliers.

An increasing trend of fluorescent signal from control samples tometastatic samples for the two biomarkers CA 15-3 and Osteopontin wasobserved. For the other two biomarkers, however, this trend is moresubtle. This assay was planned such that each slide contained allcontrol, early stage, or metastatic samples. This allowed measurement oftechnical variations within a slide only for one data set, and notacross the three cancer groups. Since this assay did not include anycontrols on the slides, it was difficult to measure and account forslide-slide variation. Therefore, an accurate measure of fluorescentresponse to disease state could not be made. Using a total of 30 patientsamples, however, showed promise for at least two of the biomarkers inas a diagnostic test. We expanded this study by including more samplesin hopes of improving the sensitivity for Her-2 and MMP-2 as well as toconfirm the results obtained for CA 15-3 and Osteopontin.

Multiplex Biomarker Study Using Patient Sera

In the breast cancer patient pilot study, the layout of the samples onthe slides made it difficult to separate the effects of slide-to-slidevariation from true differences in biomarker levels among normalsubjects and patients with early or metastatic breast cancer.Differences were seen between metastatic and control sera for CA 15-3and OPN only upon ignoring the effect of slide-slide variation. Thus, anadditional study was designed in a different manner, to account forthese variables. In this assay, antibody arrays were prepared similar tothose described above, except that two additional controls were includedon the slide. Bovine serum albumin (BSA) served as negative control(NC). Alexa 546 spots were used as positive control (PC). The samplesize was increased to 87 patient samples in this study to include 31metastatic, 23 early stage and 29 control samples. FIG. 14 shows thelayout of this experiment, which was planned such that each slideconsisted of a combination of control, early stage, and late stagesamples. The sample was split into two aliquots, assigned to two arrays,on different slides. The samples were randomly assigned to the eightwells. On the last two slides (21 and 22) blank samples were included toaccount for the background fluorescent signals. This arrangement helpedto account for the various technical variations that were not calculatedin the pilot study.

FIG. 15 shows box plot representations of the fluorescent signalsobtained for the four biomarkers as well as controls on the 22experimental slides. The positive control (Alexa-546) spot fluorescenceremained at a constant high level across all the experimental slides andthe negative control (BSA) spot fluorescence was consistently low acrossall 22 slides. An increasing trend of fluorescent signal from controlsamples to metastatic samples was observed for all of the fourbiomarkers. The multiplexed microarray assay was able to distinguishbetween control, early stage, and metastatic populations for all thebiomarkers with a fairly high accuracy (p value<0.05). Samples whichcontained only blank blocking grade human serum (slides 21 and 22) wereused to measure the biomarker background noise levels. In the FIG., thisbackground value matches that observed for the negative control (BSA)spots. NC=Negative control, O=OPN, PC=Positive control, C=CA 15-3,H=Her-2, and M=MMP-2.

Her-2 Status of Patients

Her-2 gene is amplified in about 30% of all breast cancers.HER2-positive breast cancers tend to be more aggressive than other typesof breast cancer and they are also less responsive to hormone treatment.Trastuzumab (Herceptin) is a monoclonal antibody drug that targets HER2and is used as an effective form of treatment for Her-2 positive breastcancer patients. This is shown to slow the growth of the cancer and evendecrease its size. Herceptin can be used as a treatment by itself orcombined with chemotherapy. Herceptin is also shown to reduce breastcancer recurrence by as much as 50 percent, thereby demonstrating a highrate of success in the patient survival. Detecting the Her-2 status ofbreast cancer patients, therefore, has become a routine and crucial stepin treatment decision making. The standard procedure for determiningwhether a patient is Her-2 positive involves the use of Fluorescence InSitu Hybridization (FISH) to detect the over amplified Her-2 gene.Sometimes, an ELISA is performed to measure serum Her-2 levels, in whichcase, the patient is said to be Her-2 positive if the serum Her-2 levelis higher than 15 ng/ml. The Her-2 status information was obtained forsome of the patient serum samples from the Lombardi cancer center'sserum repository and compared it to the measured Her-2 levels in sera ofthe same patients using the multiplexed microarray assay. Using the samerule as the ELISA, a patient serum sample was declared as Her-2 positiveif the Her-2 levels were measured above 15 ng/ml. The results (shown inTable 1) show a 100% correlation in the conclusions of Her-2 statusderived from our multiplexed assay and by traditional FISH or ELISAmethods (as obtained from the Lombardi Cancer Center). While both thesemethods consume large (a few hundred microliters) quantities of sampleand reagents and require anywhere between 8-24 hours to produce results,the microarray assay was performed with 50 ml of serum sample in 3hours. The value of the multiplexed assay lies in the fact that thepanel of biomarkers could not only be used for disease diagnosis, butalso to simultaneously provide valuable information about treatmentoptions for the patient.

TABLE 1 Her-2 status of sample Sample Stage of Lombardi protein Measured# Cancer Cancer Center microarray Her-2 level 1 Early stage − − 9.62 2Early stage − − 12.54 3 Early stage − − 12.38 4 Early stage − − 13.47 5Early stage − − 7.42 6 Early stage − − 5.37 7 Early stage − − 9.03 8Early stage − − 10.21 9 Early stage + + 17.44 10 Early stage + + 19.7711 Early stage + + 24.07 12 Early stage + + 23.62 13 Early stage + +19.32 14 Metastatic − − 11.62 15 Metastatic − − 13.79 16 Metastatic + +45.73 17 Metastatic + + 46.82 18 Metastatic + + 49.15 19 Metastatic + +13.43

Quantitation of Biomarker Levels in Patient Sera from FluorescentSignals

Antibody microarrays were used to generate standard curves to quantifythe biomarkers in patient serum samples. These curves were obtained in asimilar manner to the experiment described above, except that thesecurves were obtained on the same day and using the same set of slides asthe patient samples, to minimize technical error. Capture antibodies tothe four biomarkers were printed on modified microarray slides using arobotic arrayer such that each antibody was present in quadruplicate.Slides were then incubated with 8 serial dilutions of recombinantantigen diluted in human serum. The concentration ranges used were 1ng/ml-128 ng/ml for Her-2, 10 ng/ml-1280 ng/ml for MMP-2, 2 U/ml-256U/ml for CA 15-3 and 10 ng/ml-1280 ng/ml for OPN. All four detectorantibodies were used in this experiment to simulate the multiplexedassay with the patient samples. Standard curves were obtained for eachbiomarker by quantifying the fluorescence from the arrays and plottingthe median values as a function of antigen concentration. This can beseen in FIG. 16 (A-D). The standard curves were used to quantify thefluorescent intensities from patient serum samples assayed on themultiplexed arrays. This demonstrates the sensitivity and accuracy ofour multiplexed microarray assay.

Relationship Between Use of Multiple Biomarkers and Accuracy ofDiagnosis

Although box plots shown above provide information about thedistribution of the data for each individual biomarker, they are unableto provide information about all four biomarkers simultaneously. Thismethod also does not address the hypothesis that multiple markersworking in synergy provide a more sensitive diagnosis of breast cancer.Thus, Principal Component Analysis (PCA) was used to visualize the datafrom all four biomarkers in one single plot. PCA is a classicalstatistical method that reduces the dimensionality of a data set whileretaining as much information as is possible. It performs a lineartransformation that chooses a new coordinate system for the data setsuch that the greatest variance of the data set lies on the first axis(called the first principal component), the second greatest variance onthe second axis, and so on. It can be viewed as a rotation of theexisting axes to new positions in the space defined by the originalvariables. There can be as many principal components as there arevariables. The first principal component accounts for much of thevariability in the data, and each succeeding component accounts for theremaining variability. Principal components were computed for the dataset that contains fluorescence signals from four biomarkers. FIG. 17shows a scatter plot of first principal component on the X-Axis and thesecond principal component on the Y-Axis. Each vector of the principalcomponents corresponds to a patient serum sample, displayed in the FIG.as control populations in green, early stage patients in pink andmetastatic patients in blue. It can be seen that the three groups arewell separated with little or no overlap. This demonstrates that thefour biomarkers are able to distinguish accurately between controlpopulations and patients with early stage and late stage breast cancer.

For a diagnostic test, accuracy of classification is an important factorin its success. To measure the accuracy of classification of the testand to study the effect of number of biomarkers on this accuracy, LinearDiscriminant Analysis was performed on the data set. This technique isuseful for detecting the variables that allow the discrimination betweendifferent naturally occurring groups (e.g., breast cancer stages), andfor predictive classification of unknown cases into their correctgroups, establishing the robustness of this discrimination. Thisanalysis was performed by dividing the data set into two parts. Thefirst half was used as a training set and the second part was used as atest set. A linear discriminant function was built for the training setof the data and used the test set for cross-validation of predictiveclassification. This was used to produce unbiased estimates of chance ofmisclassifying the test set into the biological groups defined by thetraining set. This test was performed for all possible combinations ofthe four biomarkers to see the effect of number of biomarkers in thepanel on the accuracy of classification. FIG. 18 illustrates thepercentage of misclassifications occurred when the data was analyzedusing various combinations of biomarkers. O=OPN, C=CA 15-3, H=Her-2, andM=MMP-2. It is observed that when only one single biomarker is used, theerror of classification can be as high as 30%. When two biomarkers areused together, this error is reduced to approximately 24.35%. Addinganother biomarker further reduces this error to approximately 18.5% andfinally including all four biomarkers results in a 16.5% error. Thereduction in error of classification is drastic from one to twobiomarkers, but this difference starts to plateau when three and fourbiomarkers are included. Thus, it is possible that while multiplebiomarkers do improve the accuracy of a diagnostic test, too manymarkers, can not provide any additional information. This is however,also determined by the biological nature of the biomarker and itsphysiological role in breast cancer. It should be noted that inparticular the errors for CA 15-3 and Osteopontin are much less thanthose for Her-2 and MMP-2.

Example 3 Development of a Multiplexed Flow-Based Detector

A flow based platform was built with a unique custom flow channel deviceonto which, the multiplexed assay was translated from the proteinmicroarrays. Below, the development of the device is described. A newflow-based immunoassay system for the simultaneous and rapidquantification of multiple analytes without several processing steps isdescribed. This example also discusses the details of the device design,which is based on the standard microarray sandwich immunoassay format,except that the static incubation step was replaced with flow of theanalyte mixture over the antibody array. Multiple steps have also beeneliminated (including washing) by mixing all the assay reagents inpredetermined concentrations in one single sample. In addition, abenchtop version of a portable imaging system was developed, comprisinga miniature uncooled CCD camera and a Xenon arc lamp. This methoddemonstrated rapid quantitative measurement and specific identificationof analytes in complex samples with minimal intervention.

Materials and Methods

Microarray Flow Channel Assay and Lateral Flow Assay Components

Her-2 and Osteopontin protein, capture and biotinylated detectionantibodies as well as MMP-2-specific capture and biotinylated detectionantibodies were purchased from R&D systems (Minneapolis, Minn.). Otherreagents used in the assay include: CA 15-3 antigen and anti-CA 15-3capture and detection antibodies (Fitzgerald, Concord, Mass.), MMP-2Proenzyme (EMD Biosciences, San Diego, Calif.), biotin-BSA (PierceBiotechnology, Rockford, Ill.) and Streptavidin Quantum Dot 605,Streptavidin Quantum Dot 585, Goat-anti-mouse-Quantum Dot 605 andQuantum Dot incubation buffer (Quantum Dot Corp (Invitrogen), Hayward,Calif.) and Strepavidin Alexa 546 (Molecular Probes Invitrogen Corp.Carlsbad, Calif.). Phosphate Buffered Saline (PBS; 50 mM potassiumphosphate, 150 mM NaCl; pH 7.4) and Phosphate Buffered Saline with 0.05%Tween 20 (PBS-T) and BSA were obtained from Sigma Aldrich Corp. (St.Louis, Mo.). The CA 15-3 detection antibody was biotinylated using a kitand according to the manufacturer's instructions (Pierce Biotechnology,Rockford, Ill.). All other detection antibodies were purchased as biotinconjugates. Lyophilized human serum was purchased from RocklandImmunochemicals (Gilbertsville, Pa.). Sera from metastatic and earlystage breast cancer patients and controls were obtained from the BreastCancer Serum Biomarkers Resource, Lombardi Cancer Center (Washington,D.C.). The nitrocellulose membrane (NC) HF180, polystyrene clear backingand conjugate pad were from Millipore Corp. (Watertown, Mass., USA).GAPS II™ glass slides for the microarrays were obtained from CorningLifesciences (Corning, N.Y.) and the silicone chambers from GraceBiolabs (Bend, Oreg.). Capture antibodies used were: 1) Her-2 (R&Dsystems; Monoclonal Anti-human ErbB2 Antibody; MAB-1129; Clone 191924),2) MMP-2 (R&D systems; Monoclonal Anti-human MMP-2 Antibody; MAB-902;Clone 36006.211), 3) CA 15-3 (Fitzgerald; Monoclonal Anti-human CA 15-3Antibody; 10-C03; Clone M8071022), 4) Osteopontin (R & D Systems;Monoclonal Anti-human Osteopontin Antibody; MAB-1433; Clone 190312), and5) VEGF (Biosource; VEGF purified mouse anti-human; AHG011; CloneA183C-13G8). Detection antibodies used were: 1) Her-2 (R & D Systems;Polyclonal Goat Anti-human ErbB2 Antibody; AF-1129), 2) MMP-2 (R & DSystems; Polyclonal Goat Anti-human MMP-2 Antibody; AF-902), 3) CA 15-3(Fitzgerald; Monoclonal Anti-human CA 15-3 antibody; 10-C03B; CloneM8071021), 4) Osteopontin (R & D Systems; Polyclonal Goat Anti-humanOsteopontin Antibody; AF-1433), and 5) VEGF (Biosource; PolyclonalRabbit Anti-human VEGF Biotin Conjugated Antibody; AHG9119).

Preparation of LFA Strip

Similar to the conventional immunochromatographic strips, the LFAcomprised a nitrocellulose membrane for separation and detection ofanalytes, a conjugate pad to collect the antibody-antigen complex, awicking pad for the generation of capillary action, and a plasticbacking card for the protection of the strip (see FIG. 19).Nitrocellulose membrane, the conjugate pad, and the absorption pad werelaminated on the adhesive side of the polyester backing card. Capturemolecules were dispensed and immobilized on the nitrocellulose membraneto form the detection zone using a precision-dispenser (Bio-Dot, Irvine,Calif.). The width of the dispensing line was 0.5-1 mm and the diameterof spots was 0.5 mm (20 nl) and 0.25 mm (10 nl) with a vertical spacingof 1.5 mm and horizontal spacing of 4 mm. 5 mm strips were cut using theguillotine cutter (Bio-Dot, Irvine, Calif.) to form individual tests.

LFA Assay Protocol

We adapted the principle of immunochromatographic assay for the analysisof multiple analytes in this study. Instead of 40-nm gold particles asused in conventional immunochromatography, Quantum Dots (QD) were usedas the signaling molecule, which is bound to the detector antibodythrough a biotin-streptavidin bond. Fluorescent immunoassays wereperformed on these customized lateral flow membranes. In order todemonstrate proof of principle in the LFA system, biotin-BSA was spottedas a capture molecule onto nitrocellulose strips in both line (1 mm) andspot (20 nl and 10 nl) formats. Streptavidin-conjugated quantum dotsolution was then wicked through the biotin-BSA.

To demonstrate the principal of multiplexed LFAs, the nitrocellulosemembrane was spotted with biotin-BSA (500 μg/ml and mouse IgG μg/ml) ascapture molecules and a mixture of Streptavidin-conjugated QD 585 (10nM) solution and Goat anti mouse conjugated QD 605 (10 nM) solution wasthen wicked through the membrane. Resulting fluorescent signals due tothe accumulation of quantum dots at these sites were observed on a UVtransilluminator. For membranes that were spotted with captureantibodies, sandwich-type fluorescent immunoassays were performed oncustomized lateral flow membranes. When an analyte is first mixed withthe quantum dot-linked detector antibody and then applied to thenitrocellulose membrane, the analyte-detector antibody-quantum dotcomplex moves from the sample pad toward absorption pad. During thepropagation, the complex encounters the immobilized capture antibodieson the nitrocellulose membrane and forms sandwich-typedetector-analyte-capture antibody complexes. This fluorescence wasobserved on the UV transilluminator.

Preparation of Microarray Channels

Unique flow channels were designed that allow for simple capillary flowof assay reagents without the need for complex fluidics and mechanicalparts. Antibody microarrays were printed similar to traditionalmicroarrays using a robotic arrayer (Norgen Systems Inc.; Mountain View,Calif.). Two print heads were used to deposit approximately 1 mL ofcapture antibody solution, generating a total of 2 arrays per slide with225 μm diameter spots with a spot-to-spot distance of 350 μm. The layoutof each 8×12 array of printed antibody spots corresponded to one spotper well in a standard 8×12 (96-well) format. These capture antibodieswere printed in quadruplicate at a concentration of 1 mg/ml. Alsoprinted on each slide were two controls. Bovine serum albumin (BSA)served as negative control (NC) and Alexa 546 was used as positivecontrol (PC). The spotted slides were cross-linked under ultravioletlight for 5 minutes and were stored in the dark at 4° C. This schemeyields two channels per slide. As shown in FIG. 1, the channels arecreated by using adhesive silicone supports (0.5 mm height) and a glasscover slip facilitating fluid exchange through an inlet and an outlet(length=3 cm and width=1 cm). This helps draw fluids onto theimmobilized antibody microarrays through capillary action. The fluidflow stops at the end of the channels at which point, it can be wickedwith an absorbent material, the porosity of which can used to controlthe flow rate through the channels and hence the assay time.

Channel Assay Protocol Using Recombinant Antigen

For demonstrating proof of principle of quantitative detection on themicroarray channels, protein microarray slides were printed withbiotin-BSA in a series of 4 dilutions from 500 μg/ml to 62.5 μg/ml. Thetwo arrays on the glass slide were separated using silicone channels asdescribed above and were blocked with 1 mg/ml of BSA solution in PBS for30 minutes. This solution was wicked and the arrays were allowed to airdry for two minutes. 200 μl of 10 nM streptavidin QD 605 was drawn intothe channels and the fluid was completely wicked at the other end. Theflow channels were removed and arrays were washed and air dried prior toscanning.

To demonstrate the multiplexing capability of the channel flow assays,two different capture molecules were immobilized; biotin-BSA at 500μg/ml and mouse IgG at 500 μg/ml. Unconjugated BSA was used as anegative control. The microarrays in the channels were blocked with BSAat 1 mg/ml for 30 minutes followed by incubation with a streptavidin QD605 and Goat anti-mouse IgG—QD 605 for 2 minutes in four differentconfigurations. In the first set, no reporter was used, in the secondset, both streptavidin QD 605 and Goat anti-mouse IgG-QD 605 were; inthe third set, only streptavidin QD 605 and in the final set, only Goatanti-mouse IgG-QD 605 was used. The flow channels were removed andarrays were washed and air dried prior to scanning.

To demonstrate the sensitivity of the flow channels, standard curveswere obtained for the four biomarkers of interest using sandwichimmunoassays. A mixture of biotinylated detector antibody (at 10 μg/ml)and streptavidin-linked Alexa 546 (at 7 μg/ml) was added to serialdilutions of recombinant antigens prepared in phosphate buffered saline(PBS) and human serum. The antigen concentration ranges used were6.25-100 ng/ml for Her-2, 62.5-1000 ng/ml for MMP-2, 9.4-150 U/ml for CA15-3 and 94-1500 ng/ml for OPN. 200 μl of the sample mixture was addedto the entry of the channels. Capillary forces helped wick this fluidinto the channels over the printed capture antibodies for binding.Samples were added to duplicate arrays across different slides toaccount for technical variations. Following the appropriate incubationtime, the fluid was completely wicked and the flow channels wereremoved. The chambers were then removed and the slides were agitated inwash buffer for 5 minutes and air dried prior to imaging.

To measure the specificity of the flow channel assays, a multiplexedassay was performed essentially as described for the standard titrationcurves. Capture antibodies for Her-2, MMP-2, CA 15-3 and OPN werespotted in quadruplicate at 500 μg/ml on the GAPS II™ slides to form a4×4 array grid. A total of eight different samples were prepared, fourof which contained a mixture of all but one antigen and four of theremaining samples contained only one antigen each. The concentrations ofrecombinant antigens diluted in human serum were 20 ng/ml Her-2, 800ng/ml MMP-2, 130 U/ml CA 15-3 and 900 ng/ml OPN. Antibody microarrayswere incubated with these antigen samples mixed a detector antibody“cocktail” containing biotinylated antibodies for all five biomarkers ata concentration of 15 μg/ml. Streptavidin conjugated Alexa 546 was usedas the reporter at a concentration of 10 μg/ml. The samples were assayedin duplicate. The chambers were then removed and the slides wereagitated in PBS-T for 10 minutes and dried by centrifugation prior toscanning.

To determine the optimum speed of the assay without compromising theassay sensitivity, a multiplexed assay was performed, essentially asdescribed above. Two sets of samples were prepared. In the first case,the antigens were used at concentrations known to be present inmetastatic cancer patients (30 ng/ml for Her-2, 850 ng/ml for MMP-2, 150U/ml for CA 15-3 and 875 ng/ml for Osteopontin) and in the second case;the antigens were used at concentrations known to be present in controlpatients (8 ng/ml for Her-2, 600 ng/ml for MMP-2, U/ml for CA 15-3 and440 ng/ml for Osteopontin). 4 aliquots of 200 μl of the sample mixturewas added to duplicate arrays and incubated for 7 min, 15 min, 30 minand 60 min respectively before sample was wicked. The chambers were thenremoved and the slides were agitated in wash buffer for 5 minutes andair dried prior to imaging.

Channel Assay Protocol: Serum Samples

Two types of experiments were performed to measure the response of theflow channels to the protein biomarkers in the sera of patients withbreast cancer. In the first study, 80 μl of sera from each of the 10metastatic breast cancer patients and 80 μl of sera from each of the 10control subjects was pooled to obtain a total of 800 μl of metastaticbreast cancer sample and 800 μl of control sample. 80 μl of this pooledsample was wicked across 10 arrays in duplicate for 7 min and 15 minrespectively. The chambers were then removed and the slides wereagitated in wash buffer for 5 minutes and air dried prior to imaging.This helped estimate the technical variations in the assay. To measurethe biological variation, 80 μl of patient sera from 6 metastatic breastcancer patients and 6 control subjects was then mixed with 60 μl ofbiotinylated detector antibody cocktail and 60 μl of streptavidin Alexa546 reporter to yield a final concentration of 10 μg/ml for theantibodies and 7 μg/ml for the reporter. This sample mixture was addedto the arrays and incubated for 15 minutes before sample was wicked. Thechambers were then removed and the slides were agitated in wash bufferfor 5 minutes and air dried prior to imaging. Statistical comparison ofthe biomarker levels in breast cancer patients and controls wasundertaken using a t-test and a probability value of was obtained usingMATLAB software.

Bench Top Imaging System

As a gold standard imaging system, the ScanArray™ 5000 XL (PerkinElmer,Inc.; Wellesley, Mass.) was used at 543 nm excitation. This is abenchtop, laser-based confocal scanning device with a photomultipliertube (PMT) for sensitive fluorescence detection. Images collected onto aPC were analyzed by QuantArray™ software. Raw intensities for each spotwere computed by taking the average of the logarithm of the intensityover all pixels in the region of interest that were greater than zerofor quadruplicate spots on a slide and across duplicate chambersresulting in a total of eight spots per sample for analysis.

A imaging system was also developed that supports a charge coupleddevice (CCD) camera as this technology is more amenable to buildingsmaller portable instruments. The arrangement of this imaging system isshown in FIG. 2 and it consists of a scientific-grade 16-bit, 1392×1040pixel CCD camera (Lumenera Corp. MA), which is configured for Köhlerepi-illumination of the sample microarray. In this case, the fluorescentsample is illuminated from the front, while simultaneously being imagedfrom the same side by the CCD camera. Excitation light from a full-fieldWhite Lite® light 300 W xenon arc lamp was bandpass filtered using a 525nm excitation filter (Omega Optical Inc, Vt.) and focused uniformly onthe sample using a set of two optic fiber cables (mellesgriot) held atan angle of 45 degrees. The fluorescent spots were focused onto the CCDusing a camera lens (Infinimite® alpha, Edmund Optics) and filteredusing a 600 nm longpass filter. Custom algorithms, built within theLumenera camera software corrected for CCD dark noise. Images saved intiff format were analyzed using the Scanarray Express™ software (PerkinElmer, Wellesley, Mass.).

Results

A method was developed that requires the addition of only one fluidsample and can therefore measure multiple biomarkers simultaneously inone simple process. This technology showed a potential to facilitatecommon use of antibody microarray in medical and scientific field forhigh-throughput detection of a wide variety of analytes.

Lateral Flow Assays

To achieve rapid, multiplexed detection, an LFA (as shown in FIG. 19)was developed which combines the multiplexed, quantitative advantages ofthe protein microarrays and the assay speed and simplicity oftraditional LFA. Multiple capture molecules were spotted onto thenitrocellulose membrane, and the reporter mixture was applied to theconjugate pad at one end of the membrane. This complex was drawn throughthe membrane by capillary action, where the markers were captured bytheir respective ligands. This simple, yet rapid method required theaddition of only one fluid sample without the need for washes.Fluorescent QD nanocrystals were employed as the reporter since theyhave an added advantage of being multiplexed, yet quantitative. The useof spectrally different QDs as well as spatial separation of these twocapture molecules enables reliable multiplexed detection in this lateralflow format.

To demonstrate the proof of concept of multiplexed lateral flow assays,two test analytes (Biotin-BSA and mouse IgG) were detected on a singleLFA. As shown in FIG. 20, when a mixture containing streptavidinconjugated QD 585 and Goat anti-Mouse conjugated QD 605 (both at 10 nM)is added to the conjugate pad (left side of the FIG.), it flows throughthe pores of the nitrocellulose membrane, to the capture zones where thestreptavidin conjugated QD 585 binds to the biotinylated BSA and Goatanti-Mouse conjugated QD 605 binds to the mouse IgG respectively. Sincethe capture molecules are fixed on the membrane, the reporterscontinuously accumulate on the capture zone. This generates a signalproportional to the amount of immobilized capture molecules. FIG. 20(A)demonstrates a strip with biotin-BSA spotted as two consecutive 1 mm(100 nl) lines along the length of the nitrocellulose membrane at aconcentration of 500 μg/ml and at 125 μg/ml in FIG. 20(B). In order tomeasure multiple analytes along the strip, it is important that the flownot be obstructed by the capture molecules. It is observed that with acapture concentration of 500 μg/ml very little signal is obtained fromthe second line. However, when the concentration of the capture moleculeis reduced to 125 μg/ml, some signal is observed on the second line.When the concentration is kept high (500 μg/ml), but the dispensingvolume is reduced to 20 nl, five columns of capture molecule can beobserved (FIG. 20(C)). However, in this case, a reduction of signalintensity is observed in the direction of sample flow. When this capturevolume is reduced to 10 nl (FIG. 20(D)), this gradation disappears andthe spots have uniform fluorescence signal along the length of themembrane. The result suggested the possibility that multiple targetproteins could be detected by adjusting the amount of capture antibodyon the strip. To demonstrate this multiplexing capability of the LFA,one column (3 spots) containing biotin-BSA was immobilized and a secondcolumn (3 spots) containing mouse IgG (FIG. 20(E)). Two spectrallydistinct sets of QDs were used for this assay and the mixture containingthe two different detectors is accurately resolved on the LFAdemonstrating its multiplexing capability.

The microarray LFA was then employed for the detection of the panel ofbreast cancer biomarkers including Her-2, CA 15-3 and Osteopontin.Monoclonal capture antibodies to the four antigens were immobilized onthe nitrocellulose membrane as 10 nl spots. This spotting was done insix schemes such that the sequence in which the sample encountered thecapture molecules was different in each assay. A mixture of antigens andbiotinylated detector antibody labeled with streptavidin QD 605 asdeposited on the conjugate pad. The concentrations of reagents used inthis assay are Her-2 5 μg/ml, CA 15-3 500 U/ml and Osteopontin 3 μg/ml,biotinylated detector antibodies 30 μg/ml and strepavidin QD 605 10 nM.The solution was allowed to wick through the membrane and the spotfluorescence was observed on the UV transilluminator. Images werecaptured using a digital camera and displayed in FIG. 21, which showsthat it is possible to observe signals from three antigens in thismultiplex format. The signal to noise ratio was best for the case (C)and (E) where Her-2 is spotted at the far right hand side of themembrane. Although high concentrations of antigens were used, signal wasbarely observed above the high background in the membrane.

Accumulation of fluorescent quantum dot in the pores of thenitrocellulose membrane, as well as the high concentration of reagentsin this assay cause a high background noise in the LFA. The LFAs hadmuch lower sensitivity than that of the microarrays. The concentrationsof capture and biotinylated detector antibodies were 8 fold and 6 foldhigher than those used in the protein microarrays respectively, makingthe assay very expensive. Although the Her-2, CA 15-3 and OPN weredetected in a multiplexed format, MMP-2 assay did not produce anysignals on the LFA. The detection limits of the LFA for the antigenswere approximately 10 fold higher than protein microarrays.

The reagents for the multiplexed assay were optimized specifically forthe protein microarray platform as discussed above. By optimizing abrand new set of reagents for the LFA, it could improve the sensitivityof the assay as well as make MMP-2 work with the panel of biomarkers.The use of a membrane substrate with high affinity to proteins made LFAsprone to very high background noise. This is partially due to the factthat LFAs do not involve wash steps, and that the flow of analytesolution is through the membrane and not simply above the surface. Thisoffers a three dimensional matrix to which the analyte, detectorantibody and reporter complexes can bind non-specifically. Unlike thetraditional Western Blots, ELISAs or Microarrays, the blocking agentsemployed to minimize the background levels bind to the membrane matrixand offer resistance to the flow of analyte through membrane.Additionally, such additives can displace capture reagents from themembrane, thereby, reducing assay sensitivity.

Channel Flow Assays

To design a new platform for rapid immunoassays, the antibody arrayswere printed on a glass slide and instead of the static incubationchambers, unique flow channels were designed that cover individualarrays, and allow for passive flow of analyte mixture over theimmobilized array. Channels are made by using adhesive silicone supportsand a glass cover slip. This helps draw fluids onto the immobilizedantibody microarrays through capillary action. Fluid flow enhances thekinetic interaction between the analyte and the immobilized ligandthereby overcoming the diffusion limitation of the incubation assays andreducing assay time. Since the flow is over the arrays and not through athree dimensional matrix as in the case of LFAs, the arrays can betreated with blocking agents to minimize background noise. This providesa rapid, simple, yet multiplexed platform to measure protein biomarkersin serum samples.

A mixture containing sample, detector antibody and fluorescent reporterat predetermined concentrations was added to the protein array.Capillary forces directed this fluid into a chamber over the printedcapture antibodies for binding. The fluid was then wicked from the otherend of the channel using absorbent material. Flow rate through thesecapillary channels was controlled by choosing appropriate wickingmaterial. This also ensures unidirectional flow of the sample. Theprotein biomarkers are quantified by an optical reader as thefluorescence of the spots is proportional to the analyte concentration.This technique enabled the rapid measurement of multiple proteinbiomarkers under flow conditions. The speed of the assay offersconsiderable advantages over more conventional antibody microarrays thatrequire long incubation times.

To demonstrate the proof of principle of quantitation using thechannels, biotin-BSA was immobilized in a series of 4 dilutions from 500μg/ml to 62.5 μg/ml. Streptavidin QD at 10 nM was used as reporter. Thearrays were imaged and the fluorescent intensities of the spots wasquantified and plotted in the FIGS. 22(A and B, respectively). The spotsshow an increase in fluorescent intensity with increased concentration.A linear response was observed in this assay demonstrating the principlethat quantitative standard curves can be obtained using the microarraychannel flow device. To demonstrate the multiplexing capability, twodifferent capture molecules were used in quadruplicate (shown as columnsin the FIG. 22(C)); biotin-BSA at 500 μg/ml and mouse IgG at 500 μg/ml.BSA was used as the negative control. Both spots and were washed with amixture of streptavidin QD 605 (10 nM) and Goat anti-mouse IgG—QD 605(10 nM) for 2 min. Observed in the FIG. 22(C) are four sets of spots. Inthe first set on the left, no reporter was used and we see no signal. Inthe second set, both streptavidin QD 605 and Goat anti-mouse IgG—QD 605were used and fluorescence is observed in both the biotin-BSA and MouseIgG spots. In the third set, only streptavidin QD 605 and therefore onlythe biotin-BSA spots show fluorescence. In this final set, only Goatanti-mouse IgG—QD 605 is used resulting in fluorescence only in theMouse IgG spots. This demonstrated the specificity of the microarraychannels and its ability to measure two different analytessimultaneously.

The biotin-BSA and Mouse IgG spots were observed with “comet tails” orstreaks in the direction of flow in the channel. Since this assay wasdesigned purely for the demonstration of proof of principle, theconcentrations of the reagents were not optimized. As a result, too muchcapture molecule was deposited onto the glass surface, resulting inexcess unbound ligand, which bound to the fluorescent reporter moleculesin solutions and were smeared on the glass surface generating astreaking effect. To optimize spot morphology and minimize streaking,capture molecule concentrations should be optimized.

Flow Channel Standard Curves with Quantum Dots

Standard curves were generated on flow channels by printing captureantibodies to the four protein biomarkers such that each antibody waspresent in quadruplicate within one channel. These arrays were incubatedwith 6 serial dilutions of recombinant antigen diluted in human serum.Standard curves were obtained on the protein microarray format for eachbiomarker. FIG. 23 shows a composite image of six different arrays witheach column representing a different array that was incubated withincreasing antigen concentration from right to left. Slides shown inPanel A were incubated with Her-2 at concentrations ranging from 6.25ng/ml (right) to 100 ng/ml (left). Slides shown in Panel B wereincubated with MMP-2 with a concentration range of 62.5 ng/ml (right) to1000 ng/ml (left). Slides shown in Panel C were incubated withOsteopontin at concentrations from 94 ng/ml (right) to 1500 ng/ml (left)and those shown in Panel D were incubated with CA 15-3 at concentrationsranging from 9.4 U/ml (right)—150 U/ml (left). Channels with no antigenadded were treated as background. The results show increasedfluorescence intensity with increased protein concentration forOsteopontin and CA 15-3. The fluorescence from these spots is quantifiedusing the Scanarray software and plotted as a function of antigenconcentration in FIG. 23 (E-F) for these experiments. The standardcurves were observed to be linear for Osteopontin and CA 15-3 in theclinically-relevant ranges. However, no signal from either Her-2 orMMP-2 was observed.

To further investigate and confirm these results, a multiplex assay wasperformed in which all four protein biomarkers were analyzed in onesingle microarray. In this experiment, four identical channels wereprinted with capture antibodies to the four protein biomarkers. As shownin FIG. 24, one of these slides was incubated with only CA 15-3 antigen,the second slide was incubated with only Osteopontin antigen, the thirdslide with all antigens and the fourth with no antigen. All fourbiotinylated antibodies were used in each assay. While the results forthe assays involving OPN and CA 15-3 yielded accurate and specificsignals from the correct capture antibody spots, no signal was obtainedfrom Her-2 and MMP-2 spots in the case where all antigens were added.This indicated that the assay for Her-2 and MMP-2 was not sensitive whena rapid, one-step technique was employed.

In order to troubleshoot the failure of Her-2 and MMP-2 detection onchannels, sandwich assays on Her-2 and MMP-2 antigens were performed inthree different formats. The first assay scheme was a “wash assay” whichrepresented sequential incubation of assay reagents with washes inbetween, similar to a traditional microarray assay. The second schemecalled “long assay” involved a one-step assay, where the antigen,biotinylated detector antibody and streptavidin QD 605 were premixed andincubated on the array for 60 min. The third assay scheme titled “shortassay” was similar to the “long assay” except that the arrays wereincubated for 30 min. In FIG. 25, the results from this test for Her-2(A) and MMP-2 (B) are observed. Although the spots showed brightfluorescence for the wash assay, this signal was attenuated for the longassay in which no washes were included. This signal reached backgroundlevels for the short assay for both the antigens. The quantified signalsare observed on the plot on the right hand side. This indicates that inorder for the QD to be quantitative on the channel assays for Her-2 andMMP-2, they either need a long incubation time, or they need to be addedindividually to the arrays and cannot be pre-mixed with the sample.Therefore, Alexa 546 was adopted as the reporter for the channel assayssince it had already been optimized in the above microarray assays.

Flow Channel Assays, Standard Curves Using Alexa 546

Standard curves were performed on all four antigens using streptavidinAlexa 546 as the reporter instead of QD 605. FIG. 26 (A-D) shows thequantified fluorescence from the array spots plotted as a function ofantigen concentration. Channels with no antigen added were treated asbackground. The mixture of antigen, biotinylated antibody andstreptavidin Alexa 546 was allowed to incubate in the array in thechannels for 15 min. Standard curves were obtained for each biomarkerwhich are seen in FIG. 26 A-E. Data points for each curve represent theaverage intensities of two replicate samples (and hence eight differentspots) with a coefficient of variation of approximately 15% for allprotein biomarker curves. The standard curves for all four biomarkersappear to be linear in the clinically relevant ranges. Sensitive andlinear response is observed for all the four biomarkers including Her-2and MMP-2 in the concentration range selected. T his therefore confirmsthat Alexa 546 works well as the molecular reporter for our multiplexedassay on microarray channels.

Multiplexed Assay on the Flow Channels

Microarray flow channels were used to simultaneously detect multiplebiomarkers one single sample. In this experiment, eight identical slideswere printed with capture antibodies to the four protein biomarkersprinted in quadruplicate shown as columns in FIG. 27. O=OPN, C=CA 15-3,H=Her-2, and M=MMP-2. Four arrays were incubated with a mixture of allfour but one biomarker (A) and the other four slides were incubated withonly one antigen (B). Fluorescence was observed on the spots where thecorresponding antigens were added. Some background signal is observedfrom the spots where no corresponding antigen was added to the mixture.Since human serum was used as the medium of dilution, the non specificbinding of the serum proteins to the capture antibody spots as well asthe low, normal circulating levels of the biomarkers generate thisbackground signal from the spots even the recombinant antigen was notadded. This data therefore demonstrates specific and sensitive detectionof the four biomarkers in a multiplex format on the microarray channeldevice.

The new device platform was designed to measure multiple biomarkers andto produce rapid and reliable results in less than 15 minutes.Multiplexed assay with four different interaction times were thereforeperformed. A set of eight arrays were printed with all four captureantibodies. Four of these arrays were incubated with a mixture of allantigens in the high concentrations as observed in cancer, while theother set of four arrays was incubated with a mixture of all fourantigens in the lower concentrations as observed in normal sera. Theconcentrations of antigens used to represent metastatic cancer were 30ng/ml for Her-2, 850 ng/ml for MMP-2, 150 U/ml for CA 15-3 and 875 ng/mlfor Osteopontin and the concentrations of antigens used to representnormal sera were 8 ng/ml for Her-2, 600 ng/ml for MMP-2, 15 U/ml for CA15-3 and 440 ng/ml for Osteopontin. One array from each of the sets wasincubated for 7 min, 15 min, 30 min and 60 min respectively. The medianfluorescence intensities of the four biomarker spots were measured andare displayed in FIG. 28 (A-D). The fluorescent signal increases wereobserved with a longer incubation time for all the biomarkers. However,the difference between a 30 min incubation and 60 min incubation is notas drastic as the difference between a 7 min and 15 min incubation. Thisindicates that the assay reaches equilibrium somewhere between 30 and 60minutes. Differential signal between the high concentrations(representing metastatic disease) and low concentrations (representingnormal sera) is tabulated in FIG. 28 (E). The ratio of this differentialsignal shows that the ratio for the 7 minute incubation is below 2.0 butabove 1.5 for Her-2 and MMP-2, however this ratio is very high for CA15-3 and MMP-2. This implies that only CA 15-3 and Osteopontin areappropriate for a 7 min diagnostic test. However, all biomarkers have aratio greater than 2 for a 15 min assay making this the best compromisebetween assay speed and sensitivity

Flow Channel Patient Serum Immunoassays

To test the power of the flow channels to resolve signals from cancerversus non-cancer samples accurately, breast cancer patient serumsamples were incubated on antibody arrays. Since the serum samples werelimited, the assay could not be performed with duplicates, similar tothe protein microarrays. Therefore, this assay was designed in twoparts. First, sera from 10 metastatic breast cancer patients and 10control subjects was pooled to obtain a total of 800 μl of metastaticbreast cancer sample and 800 μl of control sample. This assay eliminatedthe patient to patient variation, but enabled the measurement oftechnical variations across various channels and slides. An 80 μlaliquot of this pooled sample (mixed with biotinylated detector antibodycocktail and streptavidin Alexa 546) was drawn across 10 replicatearrays for 15 min. The resulting fluorescent intensities obtained forthe four biomarkers are plotted in FIG. 29 (A). Significant differencesbetween metastatic and control populations are observed for all fourbiomarkers with minimal technical variations (10%).

In the second part of this study, 80 μl of patient sera from 6metastatic breast cancer patients and 6 control subjects was mixed withbiotinylated detector antibody cocktail and streptavidin Alexa 546reporter and incubated on the arrays for 15 minutes. The fluorescentsignals from the four biomarkers were quantified and the medianintensities were computed, which is shown in FIG. 29(B). A t-test wasperformed on the two sample sets (metastatic and control) for all fourbiomarkers and a p-value was generated to estimate the resolving powerof the system for accurately identifying cancer vs. non-cancer samples.The table of these p-values is listed in FIG. 29 (C). We observe thatthere is a significant difference between the signals obtained formetastatic and control samples for CA 15-3 (C) and Osteopontin (D). Thisdifference reduced for Her-2 (A) and MMP-2 (B), however, p value tableindicates that the channel assay is sensitively (p<0.05) able todistinguish between metastatic and control populations for all four.

Optical Reader

A benchtop was built of a rugged, portable fluorescence imager, whosecomponents include a miniature, megapixel CCD camera and a high powerxenon arc lamp white light generator. Arrays were exposed tobandpass-filtered excitation light from the xenon source. The resultingemitted light was bandpass-filtered and collected by the CCD camera. Thefluorescence images are exported to Scanarray™ for subsequent analysis,where background is calculated by taking into account theautofluorescence inherent to the glass slide and the non-specificbinding of fluorescence probe material in the area surrounding thetarget spots on the array. FIG. 30 shows images of microarray channelsused to obtain standard curves for all four biomarkers as captured bythe CCD based imaging system. Channels shown in FIG. 30(A) wereincubated with Her-2 at concentrations ranging from 6.25 ng/ml (right)to 100 ng/ml (left). Channels shown in Panel B were incubated with MMP-2with a concentration range of 62.5 ng/ml (right) to 1000 ng/ml (left).Channels shown in Panel C were incubated with CA 15-3 at concentrationsranging from 9.4 U/ml (right)-150 U/ml (left) and those shown in Panel Dwere incubated with Osteopontin at concentrations from 94 ng/ml (right)to 1500 ng/ml (left). Human serum was used as the diluting medium inthese assays. The results show increased fluorescence intensity withincreased protein concentration.

The fluorescence from these spots was quantified using the Scanarraysoftware and plotted as a function of antigen concentration in FIG. 30(E-H). Data points for each curve represent the average intensities ofeight replicates (background subtracted) obtained using quadruplicatespots in two replicate arrays. A linear relationship was observedbetween the concentration and fluorescent intensities for all fourbiomarkers.

FIG. 31 (A-D) shows the comparison between the CCD and PMT based imagingsystems for the quantification of dose response fluorescence. Data fromthe two methods showed a linear relationship with a correlationcoefficient (r²) of greater then 0.98, indicating that both methodsproduce similar results. This result is further supported by the resultsobtained from the multiplexed assays. The channels with arrays incubatedwith pooled patient samples from 10 metastatic and 10 controlpopulations were imaged using the CCD system and compared the results tothose obtained using the PMT. A ratio of the median fluorescenceintensities obtained for metastatic populations to the medianfluorescence intensities obtained for control populations is plotted inFIG. 31 (E) for the four biomarkers using both the CCD and PMT basedimaging systems. The results from the CCD system are very similar to theones obtained by using the PMT. A system was thus developed in which thehigh sensitivities of PMTs used in the large microarray scanners ismatched by a miniature CCD camera by controlling the excitation lightintensity and the integration time of the camera sensor.

While the invention has been particularly shown and described withreference to a preferred embodiment and various alternate embodiments,it will be understood by persons skilled in the relevant art thatvarious changes in form and details can be made therein withoutdeparting from the spirit and scope of the invention.

All references, issued patents and patent applications cited within thebody of the instant specification are hereby incorporated by referencein their entirety, for all purposes.

1. A detection device, comprising: a solid support comprising aplurality of distinct capture molecule groups, each distinct capturemolecule group comprising a plurality of capture molecules specific fora biomarker, wherein the plurality of distinct capture molecule groupsis specific for a plurality of biomarkers; a cover plate, wherein thecover plate forms an upper surface positioned above the solid support; avertical support, wherein the vertical support forms a connectionbetween the solid support and the cover plate, the connection forming atleast one channel around the capture molecule groups, and wherein thechannel comprises a first end and a second end and wherein the first endof the channel comprises an opening; and an absorbent material connectedto the second end.
 2. The device of claim 1, wherein the solid supportcomprises glass.
 3. The device of claim 1, wherein the solid supportcomprises a glass slide.
 4. The device of claim 1, wherein the capturemolecules comprise antibodies.
 5. The device of claim 1, wherein thecapture molecules are specific for biomarkers selected from the groupconsisting of: Her-2, MMP-2, CA 15-3, VEGF, and OPN.
 6. The device ofclaim 1, wherein the capture molecules are specific for biomarkersselected from the group consisting of: Her-2, MMP-2, CA 15-3, VEGF, OPN,p53, CA 125, and SER.
 7. The device of claim 1, wherein the capturemolecules are specific for Her-2, MMP-2, CA 15-3, and OPN.
 8. The deviceof claim 1, wherein the capture molecules are Clone 191924, Clone36006.211, Clone M8071022, and Clone
 190312. 9. The device of claim 1,wherein the capture molecules are blocked by a blocking agent.
 10. Thedevice of claim 1, wherein the plurality of distinct capture moleculegroups of capture molecules is arranged in an array format.
 11. Thedevice of claim 1, wherein the solid support comprises at least twocapture molecule groups, the at least two capture molecule groupscomprising identical capture molecules, and each of the at least twocapture molecule groups comprising a different number of capturemolecules.
 12. The device of claim 1, wherein the cover plate comprisesglass.
 13. The device of claim 1, wherein the cover plate comprises aglass cover slip.
 14. The device of claim 1, wherein the verticalsupport comprises adhesive silicone.
 15. The device of claim 1, whereinthe absorbent material comprises a Hi-Flow Plus Nitrocellulose MembraneHF240.
 16. The device of claim 1, comprising a glass slide comprising anarray of a plurality of distinct groups of antibodies cross-linked tothe slide, each distinct group specific for a biomarker selected fromthe group consisting of Her-2, MMP-2, CA 15-3, and OPN; a glass coverslip positioned above the solid support; and a silicone adhesiveconnection between the glass slide and the glass cover slip forming atleast one channel around the antibody groups, and wherein the channelcomprises a first open end and a second end connected to a Hi-Flow PlusNitrocellulose Membrane HF240.
 17. The device of claim 1, furthercomprising a component for detecting biomarkers bound to the solidsupport.
 18. The device of claim 17, wherein said component comprises anoptical reader and a screen for displaying output from the opticalreader.
 19. A method for determining the presence or absence of aplurality of biomarkers in a sample, comprising: acquiring a liquidmixture, wherein the mixture comprises the sample; applying the mixtureto the open first end of the at least one channel of the device of claim1; allowing the mixture to flow through the at least one channel overthe solid support; absorbing the mixture with the absorbent materialconnected to the second end; and detecting the presence of biomarkers onthe solid support, wherein presence of the biomarkers on the solidsupport indicates the presence of the biomarkers in the sample.
 20. Themethod of claim 19, wherein the device of claim 1 comprises capturemolecules comprising antibodies and the liquid mixture comprises thesample, at least one detector antibody, and at least one fluorescentreporter, and further comprising the steps of analyzing the sample withan optical reader to determine the presence or absence of the pluralityof biomarkers in the sample; and outputting the data, wherein the datacomprise the presence or absence of the plurality of biomarkers in thesample.
 21. The method of claim 19, wherein the device of claim 1comprises capture molecules specific for a plurality of biomarkersselected from the group consisting of CA 15-3, OPN, Her-2, and MMP-2.22. The method of claim 19, wherein the sample comprises human bloodserum.